Spatiotemporal delivery system embedded in 3d-printing

ABSTRACT

Provided herein is a 3D printing system and related compositions, and method of using such, that can produce a polymeric microfiber having embedded microspheres encapsulating an active agent with micron precision and high spatial and temporal resolution.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication Ser. No. 62/303,843 filed 4 Mar. 2016; U.S. ProvisionalApplication Ser. No. 62/174,232 filed 11 Jun. 2015; U.S. ProvisionalApplication Ser. No. 62/148,074 filed 15 Apr. 2015; and U.S. ProvisionalApplication Ser. No. 62/144,890 filed 8 Apr. 2015; each of which areincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant DE023583awarded by the National Institutes of Health. The government has certainrights in the invention.

MATERIAL INCORPORATED-BY-REFERENCE

Not Applicable.

BACKGROUND OF THE INVENTION

Tissue engineering provides for medical applications, biosensors,experimental drug testing, or other non-therapeutic applications.Biocompatible scaffolds are generally necessary to support growth fortissue generation to occur. While conventional scaffolds can includetransplanted cells from a subject (which can reduce immunologiccomplications), commercialization of these cell-seeded scaffolds can beproblematic with respect to ex vivo cell culture, packing and shipping,contamination, or pathogen transmission. Biomaterials that are madewithout cell transplantation may overcome these commercializationissues, but presently the design and engineering of conventionalscaffolds may not perform as well.

Various methods for conventional 3D printed scaffolds in tissueengineering and regenerative medicine can be found in, for example,Jeong 2015; Lee 2010a; Lee 2014a; Lee 2009; and Lee 2014b. As anotherexample, 3D printing processes can include selective laser sintering(SLS), stereolithography (SLA), fused deposition modeling (FDM) andbioplotting (see e.g., Bose 2013). As another example, methods for 3Dprinting scaffolds with highly porous and interconnectedmicro-architecture and reconstructing anatomical shape and dimension canbe found in, for example, Lee 2010a; Lee 2014a; Lee 2009; and Lee 2014b;and Bose 2013. Methods for 3D printed scaffolds further incorporatingdelivery of agents, growth factors, or other bioactive cues to improvetissue regeneration can be found in, for example, Lee 2010a; Akkineni2015; Moioli 2006; Poldervaart 2013; and Shim 2014a. For example, acomposite of polycaprolactone (PCL), poly(lactic-co-glycolic acid)(PLGA) and β tricalcium phosphate (β-TCP) can be constructed into amembrane by 3D printing, and bone morphogenetic protein 2 (BMP-2) can beloaded in collagen gel and delivered into the membrane's pores accordingto Shim 2014b. As another example, BMP-2 adsorbed PCL/PLGA/13-TCPmembrane can be implanted in rabbit calvaria defects and result inimproved bone healing according to Shim 2014b. Similarly, hollowcylindrical PCL/PLGA scaffolds can be fabricated by 3D printing andBMP-2 can be delivered into the hollow space via collagen or gelatin forhealing of bone segmental defect according to Shim 2014b. As anotherexample, SLA can be utilized to construct nanocomposite osteochondralscaffolds consisting of a subchondral bone layer with nanocrystallinehydroxyapatite (nHA) and a cartilage layer with transforming growthfactor β1 (TGFβ1) encapsulated in PLGA microspheres (μS) according toCastro 2015. As another example, gelatin microparticles encapsulatedwith BMP-2 can be blended with alginate, followed by bioprinting into a3D porous scaffold that in turn promotes osteogenic differentiation ofMSCs according to Poldervaart 2013. As another example, bioplotted,anatomically correct PCL-HA scaffolds with TGFβ3-collagen delivery intocartilage portion can replace entire synovial joint condyles in rabbits,followed by functional regeneration according to Lee 2010a. As anotherexample, PLGA μS encapsulated with a growth factor or various growthfactors can be incorporated on the surface of 3D printed PCLmicrostrands in different regions by applying μS-suspended ethanolthrough the scaffold's microchannels. Various types of cells,biomaterial scaffolds, and/or biochemical/physical stimulations can beutilized to replace or regenerate TMJ discs as in Ahtiainen 2013; Allen2006a; Brown 2012; Hagandora 2013; Lai 2005; and MacBarb 2013.Biological material alone without cells, including reconstituted type Icollagen templates and porcine ECM-derived scaffold can partiallyreplace TMJ disc in animal models but only resulted in modestimprovement (Brown 2012; Lai 2005). Polylactide (PLA) scaffold seededwith adipose derived stem/progenitor cells (ADSCs) supported rabbit TMJdisc healing thus preventing condylar degeneration (Ahtiainen 2013).Anisotropic disc-shaped fibrocartilage in a biconcave hydrogel withfibrochondrocytes and chondrocytes can be engineered, stimulated bychondroitinase-ABC and TGFβ1 and axial compression (MacBarb 2013). But,despite the formation of anisotropic collagen alignment, it achievedneither the mechanical properties nor heterogeneous cartilaginous matrixsimilar to those of native tissues (MacBarb 2013).

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision ofa 3D-Printing delivery system and related compositions, and method ofusing such, with micron precision and high spatial and temporalresolution.

One aspect of the present disclosure provides a method of forming abiocompatible scaffold.

One embodiment provides a method of forming a biocompatible scaffold. Insome embodiments, the method includes encapsulating at least one agentin a plurality of microspheres; combining the plurality of microspheresand a matrix material, the matrix material being suitable for forming ascaffold via 3D printing; introducing the combination of microspheresand matrix material into a first cartridge of a 3D printing device;heating the combination of microspheres and matrix material in the firstcartridge sufficiently to allow dispensing of the combination whilepreventing substantial degradation of the microsphere or the at leastone agent encapsulated in the microsphere; dispensing the heatedcombination of microspheres and matrix material from the first cartridgethrough a printing needle to form a polymeric microfiber, wherein themicrospheres are distributed through the polymeric microfiber; orforming a scaffold comprising a plurality of the polymeric microfibers,wherein the microspheres are distributed through the scaffold by way ofthe polymeric microfibers.

Method features discussed above can be combined with other featuresdiscussed below.

Another aspect provides a method of forming a polymeric fiber having amicroencapsulated agent distributed in the polymeric fiber.

One embodiment provides a method of forming a polymeric fiber having amicroencapsulated agent distributed in the polymeric fiber includingencapsulating at least one agent in a plurality of microspheres;combining the plurality of microspheres and a matrix material, thematrix material being suitable for forming a scaffold via 3D printing;introducing the combination of microspheres and matrix material into afirst cartridge of a 3D printing device; heating the combination ofmicrospheres and matrix material in the first cartridge sufficiently toallow dispensing of the combination of microspheres and matrix materialwhile preventing substantial degradation of the microsphere or the agentencapsulated in the microsphere; or dispensing the heated combination ofmicrospheres and matrix material from the cartridge through a printingneedle to form a polymeric microfiber, wherein the microspheres aredistributed through the polymeric microfiber.

Method features discussed above can be combined with other featuresdiscussed above and below.

Another aspect provides a composition including a polymeric microfiberproduced by 3D printing.

One embodiment provides a composition including a polymeric microfiberproduced by 3D printing or a plurality of microspheres encapsulating atleast one agent; wherein the microsphere encapsulated agent isdistributed through the polymeric microfiber.

Features related to the composition can be combined with other featuresdiscussed above with respect to above methods of forming a scaffold orpolymeric fiber. Composition features discussed above can be combinedwith other features discussed below.

In some embodiments, the microspheres include at least a first group ofmicrospheres and a second group of microspheres. For example, the firstgroup of microspheres and the second group of microspheres can includeat least one agent. As another example, the first group of microspheresand the second group of microspheres comprise at least one differentagent. Features related to microspheres can be combined with otherfeatures discussed above and below

In some embodiments, the method or composition includes introducing thecombination of microspheres and matrix material into a second cartridgeof a 3D printing device. In some embodiments, the method or compositionincludes heating the combination of microspheres and matrix material inthe second cartridge sufficiently to allow dispensing of the combinationof microspheres and matrix material while preventing substantialdegradation of the microsphere or the agent encapsulated in themicrosphere. In some embodiments, the method or composition includesinterchanging the first cartridge and the second cartridge during aprinting process. Features related to the combination of microspheresand matrix material can be combined with other features discussed aboveand below.

In some embodiments, the method or composition includes at least oneagent, wherein the at least one agent includes a growth factor. Featuresrelated to the agents and growth factors can be combined with otherfeatures discussed above and below

In some embodiments, the method or composition includes progenitorcells, such as stem cells, wherein the growth factor stimulatesfibroblastic, chondrogenic, or osteogenic differentiation of the stemcells. Features related to progenitor cells can be combined with otherfeatures discussed above and below

In some embodiments, the method or composition includes a first growthfactor and a second growth factor alternately embedded in themicrofibers. In some embodiments, the method or composition includes atleast one agent, wherein the at least one agent includes a growth factorselected from the group consisting of CTGF, TGFβ, TGFβ3, CTGF, BMPs,SDF, bFGF, IGF, GDF, PDGF, VEGF, or EGF, or an isoform thereof. Featuresrelated to the growth factors can be combined with other featuresdiscussed above and below.

In some embodiments, the method or composition includes a matrixmaterial and a polymeric microfiber, wherein the matrix materialcomprises polycaprolactone (PCL) and the polymeric microfiber comprisesPCL. Features related to matrix material can be combined with otherfeatures discussed above and below.

In some embodiments, the method or composition includes heating thecombination of microspheres and matrix material to less than the meltingpoint of the microsphere. In some embodiments, the method or compositionincludes heating the combination of microspheres and matrix material toabout 60° C., about 65° C., about 70° C., about 75° C., about 80° C.,about 85° C., about 90° C., about 95° C., about 100° C., about 105° C.,about 110° C., about 120° C., about 130° C., about 140° C., about 150°C., about 160° C., about 170° C., about 180° C., or about 190° C.Features related to heating can be combined with other featuresdiscussed above and below.

In some embodiments, the method or composition includes encapsulatedgrowth factors, wherein the bioactivity of the encapsulated growthfactors is substantially maintained. Features related to encapsulationcan be combined with other features discussed above and below.

In some embodiments, the method or composition includes a printingneedle, wherein the printing needle can have an inner diameter of about20 μm to about 750 μm. For example, the printing needle can an innerdiameter of about 50 μm to about 400 μm. Features related to theprinting needle can be combined with other features discussed above andbelow.

In some embodiments, the method or composition includes a 3D printedscaffold including microstrands having a microstrand diameter of about100 μm to about 400 μm. In some embodiments, the 3D printed scaffoldcomprises microstrands having an inter-microstrand spacing ormicrochannel width of about 100 μm to about 600 μm. Features related tothe microstrand diameter can be combined with other features discussedabove and below.

In some embodiments, the method or composition includes a growth factorencapsulated microsphere. In some embodiments, the growth factorencapsulated microsphere has a diameter of about 10 μm to about 600 μm.In some embodiments, the growth factor encapsulated microsphere isembedded at about 10 mg to about 100 mg μS per about 1 g of matrixmaterial. In some embodiments, the growth factor encapsulatedmicrosphere has sustained release of growth factor for at least 42 days.In some embodiments, the growth factor encapsulated microsphere is about50 mg μS per 1 g matrix material. Features related to microspheres canbe combined with other features discussed above and below.

In some embodiments, the method or composition includes treating thescaffold, composition, or polymeric fiber with NaOH to createmicro-pores. Features related to NaOH treatment or micro-pores can becombined with other features discussed above and below.

In some embodiments, the method or composition includes the formation ofa multi-tissue complex and the mechanical properties or composition ofthe resulting regenerated multi-tissue complex have substantiallysimilar mechanical properties or composition of a corresponding nativemulti-tissue complex. Features related to multi-tissue complex ormechanical properties can be combined with other features discussedabove and below.

In some embodiments, the method or composition further includesdispensing a plurality of heated matrix materials or a plurality ofcombinations heated matrix materials or microspheres from a plurality ofcartridges, the contents of each cartridge independently selected. Insome embodiments, each cartridge of the plurality of cartridges includesa printing needle or a heating element. In some embodiments, eachcartridge of the plurality of cartridges shares a printing needle or aheating element. In some embodiments, a first portion of cartridges eachincludes a printing needle or a heating element and a second portion ofcartridges shares a printing needle or a heating element.

In some embodiments, the plurality of cartridges includes one or moreactive cartridges dispensing matrix material, microspheres, or acombination thereof. In some embodiments, the one or more activecartridges can be switched among the plurality of cartridges before,during, or after dispensing the heated combination.

Features related to cartridges, plurality of cartridges, or switching ofcartridges can be combined with other features discussed above andbelow.

In some embodiments, the heating temperature is about the meltingtemperature of the matrix material and the combination of the matrixmaterial and microencapsulated active agent is heated such that 90%,80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the total volume ofmicrospheres do not reach more than 60° C. for more than about 10-40minutes (e.g., about 30 minutes). In some embodiments, the combinationof the matrix material and microencapsulated active agent is heated suchthat 80% of the total volume of microspheres do not reach more than 45°C. for more than about 30 minutes. In some embodiments, a first printingcartridge comprising the combination of the matrix material andmicroencapsulated active agent is switched for a second printingcartridge comprising the combination of the matrix material andmicroencapsulated active agent before 80% of the total volume ofmicrospheres do reach more than 45° C. for more than about 30 minutes.

Features related to heating temperature, melting point, temperaturethreshold for percentage of total volume of microspheres, or heating canbe combined with other features discussed above and below.

Another aspect provides for a method of treating a tissue defect withthe composition or scaffold produced according to the features discussedabove. In some embodiments, the method includes implanting the scaffoldinto a subject in need thereof. For example, the tissue defect can beassociated with a multi-tissue interface selected from the groupconsisting of musculoskeletal system; craniofacial system; periodontium;cementum (CM)-periodontal ligament (PDL)-alveolar bone (AB) complex;ligament-to-bone insertion; tendon-to-bone insertion; rotator cuff;supraspinatus tendon-to-bone interface; interface between tendon,fibrocartilage, or bone; supraspinatus tendon-fibrocartilage-boneinterface; articular cartilage-to-bone junction; anterior cruciateligament (ACL)-to-bone complex; anterior cruciateligament-fibrocartilage-bone interface; intervertebral disc; nucleuspulposus-annulus fibrosus-endplates; cementum-periodontalligament-alveolar bone; muscle-to-tendon; inhomogeneous or anisotropictissues; knee meniscus; temporomandibular joint disc; periodontium;root-periodontium complex; synovial joints; or fibrocartilaginoustissues. Features related to methods of treatment can be combined withother features discussed above.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1A-FIG. 1F are a series of illustrations, images, and a plotshowing a spatiotemporal delivery system embedded in 3D-printedscaffold. Selected growth factors (GF) are encapsulated inpoly(lactic-co-glycolic acids) (PLGA) microspheres (μS) for controlledrelease. Further details regarding methodology and results are providedin Example 1, Example 5, Example 6, and Example 7. Scale=100 μm. Furtherdetails regarding methodology and results are provided in Example 1 andExample 5.

FIG. 1A shows a two cartridge spatiotemporal delivery system wheregrowth factors (GFs) are encapsulated in PLGA microspheres (μS) forcontrolled release. PCL and GF-encapsulated μS are mixed in dispensingcartridges of 3D Bioplotter® and heated up to 120° C.

FIG. 1B shows use of use of fluorescent dextran/μS to confirm that PLGAμS were successfully embedded in the 3D-deposited PCL microstrands (seee.g., FIG. 1B). Further details regarding methodology and results areprovided in Example 1 and Example 5.

FIG. 1C is a fluorescence image of whole scaffolds showing evenlydistributed microspheres in the 3D printed structure. Further detailsregarding methodology and results are provided in Example 1 and Example5.

FIG. 1D is a fluorescence image of whole scaffolds showing evenlydistributed microspheres in the 3D printed structure. Using fluorescentdextran/μS, it was confirmed that PLGA μS were successfully embedded inthe 3D-deposited PCL microstrands, with custom designed scaffoldstructure/pattern. Further details regarding methodology and results areprovided in Example 5.

FIG. 1E is a plot of CTGF, TGFβ3, and BMP-2 delivered in PCL scaffoldsvia μS embedding showing a sustained release up to 42 days in vitro. Theencapsulated CTGF, TGFβ3, and BMP-2 in μS embedded PCL scaffold showssustained release over 42 days incubation in vitro. Further detailsregarding methodology and results are provided in Example 5.

FIG. 1F shows the encapsulated growth factor in μS-embedded PCL scaffoldhad sustained release over 42 days incubation in vitro as shown in arepresentative data. Further details regarding methodology and resultsare provided in Example 1 and Example 5.

FIG. 1G is a fluorescence image of spatiotemporal delivery of multiplegrowth factors in 3D printed scaffolds.

FIG. 2 is a line plot showing estimated temperature distribution withinPLGA microspheres upon heating surrounding PCL from 25° C. to 100° C.The heat conduction governing equation (eq. 1) was used for thecalculation. Temperature as a function of r (radial position) and t(time) is plotted from 0 to 360 minutes at 10 different depths (surfaceto core), where R is the radius of microspheres. Given the averagefabrication time for a scaffold (−30 mins), temperature over 80% oftotal volume of microspheres is lower than 45° C., which can preservebioactivity of encapsulated growth factors. Further details regardingmethodology and results are provided in Example 1 and Example 7.

FIG. 3A through FIG. 3C are a series of images showing formation ofmulti-tissue interfaces in 3D-printed scaffold with spatiotemporaldelivered CTGF and TGFβ3 via PLGA microspheres. FIG. 3A shows a singlelayered rectangular structure. FIG. 3B shows a single layeredrectangular structure constructed with alternative PCLmicrofibers-embedding CTGF-μS and TGF TGFβ3-μS. The size of microfibersand interfibers space was 100 μm. FIG. 3C shows integrated interfacebetween COL-I+ and COL-II+ matrices was successfully formed within the˜100 μm spaces. Alternative depositions of COL-I+ and COL-II+ matriceswere formed after 4 weeks in vitro culture of hMSCs matching the patternof GF delivery within ˜100 μm spaces. Scale=200 μm. Further detailsregarding methodology and results are provided in Example 1, Example 2,and Example 7.

FIG. 4A through FIG. 4D show projected scaffold design withspatiotemporal delivery of growth factors. FIG. 4A shows projectedreconstruction of anterior cruciate ligament-fibrocartilage-boneinterfaces. FIG. 4B shows projected reconstruction of (supraspinatustendon-fibrocartilage (unmineralized and mineralized)-bone interfaces.FIG. 4C shows projected reconstruction of cementum (CM)-periodontalligament (PDL)-alveolar bone (AB) complex. FIG. 4D shows projectedrecapitulating the gradient matrix distribution and organization ininhomogeneous multiphase tissues, such as TMJ disc and knee meniscus.Histology was adopted from Lu and Thomopoulos 2013 Annu Rev Biomed Eng.15, 201-226. Further details regarding methodology and results areprovided in Example 3.

FIG. 5A through FIG. 5C are a pair of photographs showing a series ofimages of a TMJ disk scaffold. FIG. 5A and FIG. 5B show views of the TMJdisk scaffold. FIG. 5C shows the TMJ disk scaffold annotated forlocation in which CTGF, CTGF+TGFβ3, and BMP-2 was embedded.

FIG. 6 is an illustration showing a rotator cuff scaffold fabricatedwith strands having embedded CTGF, CTGF+TGFβ3, and BMP-2 along withlocation for implantation.

FIG. 7A-FIG. 7F are a series of images and graphs showing preparation ofPLGA μS and characterization of GF/μS-embedded PCL scaffold and effectsof NaOH treatment on GF release. Further details regarding methodologyand results are provided in Example 5.

FIG. 7A is an SEM image of PLGA microspheres (μS).

FIG. 7B is an SEM image of PLGA microspheres (μS).

FIG. 7C is a histogram showing the μS diameter size distribution (mean:22.68±14.89 μm).

FIG. 7D is a graph showing the mechanical properties of the 3D printedscaffolds, including compressive modulus and ultimate strength, were notsignificantly altered by embedding 50 mg PLGA μS per 1 g in PCL.

FIG. 7E is an image showing effect of NaOH treatment and effect ongrowth factor (GF) release behavior.

FIG. 7F is graph showing effect of NaOH treatment and effect on growthfactor (GF) release behavior.

FIG. 8A-FIG. 8L are a series of images showing fibrogenic, chondrogenic,and osteogenic differentiation of MSCs in GF/μS embedded 3D printed PCLscaffolds. Further details regarding methodology and results areprovided in Example 5.

FIG. 8A is a Picrosirius Red (PR) stained section of the 3D printed PCLscaffold after 4 weeks culture with MSCs (−GF).

FIG. 8B is a Picrosirius Red (PR) stained section of the 3D printed PCLscaffold after 4 weeks culture with MSCs (+CTGF).

FIG. 8C is a Picrosirius Red (PR) stained section of the 3D printed PCLscaffold after 4 weeks culture with MSCs (+TGFβ3).

FIG. 8D is a Picrosirius Red (PR) stained section of the 3D printed PCLscaffold after 4 weeks culture with MSCs (+BMP2).

FIG. 8E is a Safranin O (Saf O) stained section of the 3D printed PCLscaffold after 4 weeks culture with MSCs (−GF).

FIG. 8F is a Safranin O (Saf O) stained section of the 3D printed PCLscaffold after 4 weeks culture with MSCs (+CTGF).

FIG. 8G is a Safranin O (Saf O) stained section of the 3D printed PCLscaffold after 4 weeks culture with MSCs (+TGFβ3).

FIG. 8H is a Safranin O (Saf O) stained section of the 3D printed PCLscaffold after 4 weeks culture with MSCs (+BMP2).

FIG. 8I is a Alizarin Red stained section of the 3D printed PCL scaffoldafter 4 weeks culture with MSCs (−GF).

FIG. 8J is a Alizarin Red stained section of the 3D printed PCL scaffoldafter 4 weeks culture with MSCs (+CTGF).

FIG. 8K is a Alizarin Red stained section of the 3D printed PCL scaffoldafter 4 weeks culture with MSCs (+TGFβ3).

FIG. 8L is a Alizarin Red stained section of the 3D printed PCL scaffoldafter 4 weeks culture with MSCs (+BMP2).

FIG. 9A-FIG. 9E are a series of images showing the formation ofmulti-tissue interfaces in a 3D-printed scaffold with spatiotemporaldelivery of CTGF and TGFβ3 via PLGA microspheres. Further detailsregarding methodology and results are provided in Example 5.

FIG. 9A is an image of a single layered rectangular scaffold (1.5×1.5mm) fabricated with parallel oriented PCL microfibers (100 μm) andinter-fibers channels (100 μm).

FIG. 9B is an image showing the PCL microfibers alternatingly embeddedwith CTGF and TGFβ3 μS.

FIG. 9C is an immunofluorescence image showing integrated interfaces ofCOL-I+ and COL-II+ matrices within the 100 μm microchannels with MSCs atweeks corresponding to the alternatingly located CTGF and TGFβ3 in FIG.9B.

FIG. 9D is an enlarged section of FIG. 9C showing integrated interfacesof COL-I+ and COL-II+ matrices within the 100 μm microchannels with MSCsat weeks corresponding to the alternatingly located CTGF and TGFβ3 inFIG. 9B.

FIG. 9E is a series of histology images showing integrated interfaces ofCOL-I+ and COL-II+ matrices within the 100 μm microchannels with MSCs atweeks corresponding to the alternatingly located CTGF and TGFβ3 in FIG.9B.

FIG. 10A-FIG. 10C are a series of images showing in vitro tissueengineering of rabbit TMJ disc. Picrosirius red (PR) and Alcian blue(AB) staining of whole sections of rabbit TMJ disc show anisotropiccollagen orientation and regionally distributed cartilaginous matrix(see e.g., FIG. 10A). Collagen was distributed throughout the disc witha circumferential orientation in the posterior and anterior bands, andan anteroposterior orientation 556 in the intermediate zone. AB+cartilaginous matrix was predominantly located in the intermediate zonewith rounded chondrocyte-like cells (see e.g., FIG. 10A). From theanatomical contour of native rabbit TMJ disc, 3D printed scaffolds werefabricated with the anisotropic alignment of microstrands and deliveryof CTGF and TGFβ3 μS mimicking the regionally distributedfibrocartilaginous matrix (see e.g., FIG. 10B). After 6 weeks culturewith MSCs, CTGF/TGFβ3 μS-embedded scaffolds formed densely orientedfibrous tissue in the posterior and anterior bands, whereasfibrocartilaginous tissue in the intermediate zone, reminiscent ofnative tissue (see e.g., FIG. 10C). Further details regardingmethodology and results are provided in Example 5.

FIG. 10A is a series of images showing Picrosirius red (PR) and Alcianblue (AB) stained sections demonstrating the anisotropic collagenorientation and regionally variant fibrocartilaginous matrix in nativerabbit TMJ discs.

FIG. 10B is an image of an anatomically correct rabbit TMJ disc scaffoldfabricated with 100 μm PCL strands and inter-strands microchannels.

FIG. 10C is a series of images showing Picrosirius red (PR) and Alcianblue (AB) stained sections after 6 weeks culture with MSCs either fromhuman bone marrow or rabbit TMJ synovium. Multiphase fibrocartilaginoustissue with densely aligned fibrous matrix was observed in the anteriorand posterior bands and fibrocartilaginous matrix in the intermediatezone.

FIG. 11A-FIG. 11B are a series of images showing in vivo healing ofrabbit TMJ disc by 3D printed scaffolds. Upon performing 2.5 mm discperforation, 3D printed scaffolds (with or without GFs) were implanted.After 4 weeks, vertical tissue sections with H&E, PR, Saf-O, and ABstaining showed that CTGF/TGFβ3 μS-embedded scaffolds led to a fullrecovery of perforated disc with multiphase fibrocartilaginous tissuesimilar to native TMJ disc, whereas empty μS-embedded scaffolds resultedin severe disc degeneration with loss of fibrocartilage (see e.g., FIG.11A). In addition, healed TMJ disc with CTGF/TGFβ3 μS-embedded scaffoldsrestored rounded chondrocyte-like cell phenotype on the disc surface,reminiscent of native (see e.g., FIG. 11B). In contrast, degeneratingdisc with empty μS show a loss of chondrocyte-like cells (see e.g., FIG.11B). Further details regarding methodology and results are provided inExample 5.

FIG. 11A is a series of images showing H&E, Picrosirius red (PR), Saf-O,and Alcian blue (AB) stained sections of 3D printed μS-embedded TMJscaffold, in vivo, after 6 weeks implantation in Native TMJ, μS-embeddedscaffold+GF, and μS-embedded scaffold−GF.

FIG. 11B is an image of high magnification histology showing the roundedchondrocyte-like cell population on the surface of after 6 weeksimplantation in Native TMJ, μS-embedded scaffold+GF, and μS-embeddedscaffold−GF.

FIG. 12A-FIG. 12E are a series of images and bar graphs evaluatingarthritic changes on mandibular condyle. Gross images showed nonoticeable damage or structural changes on the articular cartilage ofmandibular condyle (see e.g., FIG. 12A). No sign of cartilage defects onthe articular surface of TMJ condyle were observed after 4 weeksimplantation of GF/μS-embedded scaffolds, whereas scaffold withoutgrowth factor resulted in some vertical erosions in cartilage, ascompared to native articular cartilage (see e.g., FIG. 12B-FIG. 12C).Quantitatively, the cartilage thickness without GF was significantlythinner than native and the GF/μS-embedded scaffolds (see e.g., FIG.12D) (*: p<0.05; n=5 per group). Consistently, OARSI osteoarthritisscore was significantly lower with GF/μS-embedded scaffolds thanscaffolds without GF (see e.g., FIG. 12E) (*: p<0.05; n=5 per group).Further details regarding methodology and results are provided inExample 5.

FIG. 12A is a series of images of resected native TMJ condyle,μS-embedded scaffold+GF TMJ condyle, and μS-embedded scaffold−GF TMJcondyle.

FIG. 12B is a series of images showing H&E stained sections of nativeTMJ condyle, μS-embedded scaffold+GF TMJ condyle, and μS-embeddedscaffold−GF TMJ condyle.

FIG. 12C is a series of images showing Safranin-O stained sections ofnative TMJ condyle, μS-embedded scaffold+GF TMJ condyle, and μS-embeddedscaffold−GF TMJ condyle.

FIG. 12D is a bar graph showing cartilage thickness with no growthfactor (−GF) was significantly thinner than native and theGF/μS-embedded scaffolds.

FIG. 12E is a bar graph showing OARSI osteoarthritis score wassignificantly lower with GF/μS-embedded scaffolds than scaffolds withoutGF.

FIG. 13A-FIG. 13C are a series of images showing the glenoid fossa after4 weeks scaffold implantation. Gross images show no noticeable cartilagedefect or structural changes in the glenoid fossa (see e.g., FIG. 13A).Histologically, a denser Saf-O positive cartilage layer was observedwith GF/μS-embedded scaffolds as compared to the native with nonoticeable sign of cartilage defects (see e.g., FIG. 13B-FIG. 13C).Further details regarding methodology and results are provided inExample 5.

FIG. 13A is a series of images of the of the native glenoid fossa,μS-embedded scaffold+GF glenoid fossa, and μS-embedded scaffold−GFglenoid fossa of the TMJ condyle.

FIG. 13B is a series of images of H&E stained sections of the nativeglenoid fossa, μS-embedded scaffold+GF glenoid fossa, and μS-embeddedscaffold−GF glenoid fossa of the TMJ condyle.

FIG. 13C is a series of images of Safranin-O stained sections of thenative glenoid fossa, μS-embedded scaffold+GF glenoid fossa, andμS-embedded scaffold−GF glenoid fossa of the TMJ condyle.

FIG. 14A-FIG. 14I are a series of images and graphs characterizing a TMJdisc scaffold. Further details regarding methodology and results areprovided in Example 6.

FIG. 14A is an image of the 3D model for a 3D-printed TMJ disc scaffold.

FIG. 14B is an image of a TMJ disc scaffold constructed with repeatingmicrostrands and interstrand microchannels with their orientationpredominantly in the circumferential and anteroposterior directions inthe peripheral ring and intermediate zone, respectively mimicking nativeanisotropic collagen alignment.

FIG. 14C is a bar graph showing the size of PCL microstrands (300 μm)and microchannels (300 μm) and the relative density of microstrandsparallel to versus perpendicular to the alignment direction (2:1) andwere determined to closely approximate the tensile properties of thoseof native disc in the circumferential and anteroposterior directions,respectively.

FIG. 14D is a representative fluorescence image with μS-encapsulatingAlex Fluora® 488 and 546 demonstrating that CTGF is delivered throughoutthe scaffolds whereas CTGF and TGFβ3 are delivered in the intermediatezone of the TMJ disc scaffolds.

FIG. 14E is a graph showing CTGF and TGFβ3 delivered in the scaffoldshowed a sustained release up to 42 days in vitro.

FIG. 14F is an image of a TMJ disc scaffold cultured with MSCs (2M/mL)for 6 weeks without μS.

FIG. 14G is an image of a TMJ disc scaffold cultured with MSCs (2M/mL)for 6 weeks with spatiotemporal delivery of CTGF and TGFβ3 in μS.

FIG. 14H is a bar graph showing collagen content per wet weight wassignificantly higher both in the intermediate zone (IZ) and theanterior/posterior band (AP) with GF delivery as compared to controlwithout GF (p<0.05; n=5 per group).

FIG. 14I is a bar graph showing GAGs content was significantly higherwith GF delivery as compared to control (see e.g., FIG. 14I) (p<0.05;n=5 per group). IZ showed significantly higher GAGs content than AP(p<0.05; n=5 per group).

FIG. 15A is a series of PR and Saf-O stained sections of TMJ discshowing, after 6 weeks culture with MSCs, growth factor (GF; CTGF andTGFβ3) encapsulated μS-embedded scaffolds formed heterogeneousfibrocartilage featured by Saf-O-positive cartilaginous matrix in theintermediate zone and PR-positive denser collagenous tissue in the APbands. The high dose (100 mg μS/g PCL) showed likely densercartilaginous matrix in the intermediate zone as compared to low dose(50 mg μS/g PCL). Further details regarding methodology and results areprovided in Example 6.

FIG. 15B is a series of higher magnification images of PR and Saf-Ostained sections of the AP band and intermediate zone with and withoutGF showing higher density of collagenous tissue in the AP bands andfibrocartilaginous tissue in the intermediate zone with GF/μS-embeddedscaffolds, in comparison to empty μS-embedded scaffolds.

FIG. 15C is a bar graph showing total collagen content in the AP bandswere significantly higher with the high dose as compared to low dose ofGF/μS and empty/μS (p<0.05, n=5 per group).

FIG. 15D is a bar graph showing total GAGs content in the IZ weresignificantly higher with the high dose as compared to the low dose ofGF/μS and empty/μS.

FIG. 16A is a series of immunofluorescence images showing that both highand low doses of GF/μS resulted in COL-II+/AGC+ fibrocartilaginoustissue in the intermediate zone, not in the AP bands. No COL-II and AGCwere found with the empty μS. Consistently, denser COL-I+ matrix wasformed with GF/μS both in high and low doses in the AP bands, incomparison with the empty μS. Further details regarding methodology andresults are provided in Example 6.

FIG. 16B is a bar graph showing relative areas positive for COL-II andAGC were significantly wider in for the high dose as compared to the lowdose. Further details regarding methodology and results are provided inExample 6.

FIG. 16C is a bar graph showing qRT-PCR showed significantly more COL-ImRNA expression with the GF/μS for high and low doses as compared toempty μS. Further details regarding methodology and results are providedin Example 6.

FIG. 17A is a bar graph showing no statistically significant differencein the tensile modulus to the direction of PCL microstrand alignmentbetween GF/μS and empty μS after 6 weeks culture with MSCs. Furtherdetails regarding methodology and results are provided in Example 6.

FIG. 17B is a bar graph showing the compressive modulus wassignificantly higher in the high dose (100 mg μS/g PCL) empty μS thanthe low dose (50 mg μS/g PCL) in the AP bands. Further details regardingmethodology and results are provided in Example 6.

FIG. 17C is a bar graph showing the compressive modulus wassignificantly higher in the high dose (100 mg μS/g PCL) empty μS thanthe low dose (50 mg μS/g PCL) in the Intermediate Zone (IZ). Furtherdetails regarding methodology and results are provided in Example 6.

FIG. 18A is a bar graph showing instantaneous (Ei) were significantlylower in high dose GF/μS than empty μS in the AP band. Further detailsregarding methodology and results are provided in Example 6.

FIG. 18B is a bar graph showing relaxation moduli (Er) weresignificantly lower in high dose GF/μS than empty μS both in the APband. Further details regarding methodology and results are provided inExample 6.

FIG. 18C is a bar graph showing the ratio of Er to Ei was significantlysmaller in the high dose GF/μS as compared to all the other groups, moreapproximating the native property. Further details regarding methodologyand results are provided in Example 6.

FIG. 18D is a bar graph showing the coefficient of viscosity (μ) wassignificantly higher with GF/μS as compared to empty μS both in APbands. Further details regarding methodology and results are provided inExample 6.

FIG. 18E is a bar graph showing is a bar graph showing instantaneous(Ei) were significantly lower in high dose GF/μS than empty μS in theintermediate zone. Further details regarding methodology and results areprovided in Example 6.

FIG. 18F is a bar graph showing is a bar graph showing relaxation moduli(Er) were significantly lower in high dose GF/μS than empty μS both inthe intermediate zone. Further details regarding methodology and resultsare provided in Example 6.

FIG. 18G is a bar graph showing is a bar graph showing the ratio of Erto Ei was significantly smaller in the high dose GF/μS as compared toall the other groups, more approximating the native property. Furtherdetails regarding methodology and results are provided in Example 6.

FIG. 18H is a bar graph showing is a bar graph showing the coefficientof viscosity (μ) was significantly higher with GF/μS as compared toempty μS both in the intermediate zone. Further details regardingmethodology and results are provided in Example 6.

FIG. 19A is a bar graph showing that on an age-adjusted basis,musculoskeletal conditions are reported by 54 persons per every 100 inthe population in US.

FIG. 19B is an illustration of a rotator cuff tear.

FIG. 20 is a series of illustrations and images showing rotator cuff andligament-bone scaffolds. Scaffold design for bone to tendon integratedmulti-tissue formation. Application for rotator cuff repair graft withthree layers 3D printed PCL/PGLA μS: PCL/CTGF+TGFβ3 μS layer sandwichedbetween PCL/CTGF μS and PCL/BMP2 μS. Further details regardingmethodology and results are provided in Example 4 and Example 7.

FIG. 21 is a series of fluorescence images of tendon-bone scaffolds at 6weeks with hMSCs (2 M/mL). +GF group induced higher collagen Iexpression than the −GF group. Further details regarding methodology andresults are provided in Example 7.

FIG. 22A-FIG. 22C are a series of microscopy images of tendon-bonescaffolds at 6 weeks with hMSCs (2 M/mL) with and without GF and a bargraph of total collagen of tendon-bone scaffolds at 6 weeks with hMSCs(2 M/mL) with and without GF. +GF group induced higher collagen Iexpression than the −GF group. Further details regarding methodology andresults are provided in Example 7.

FIG. 22A is an H&E stained section of tendon-bone scaffolds at 6 weekswith hMSCs (2 M/mL) with and without GF.

FIG. 22B is a Trichrome Blue stained section of tendon-bone scaffolds at6 weeks with hMSCs (2 M/mL) with and without GF.

FIG. 22C shows +GF group induced higher collagen I expression than the−GF group.

FIG. 23 is a series of fluorescence images of Col-II & Aggrecan at 6weeks with hMSCs. −GF group did not show any aggrecan (AGC) expressionin the ECM. Further details regarding methodology and results areprovided in Example 7.

FIG. 24 is a series of fluorescence images of osteocalcin expression at6 weeks with hMSCs with and without GF. Only the BMP2 layer in the +GFgroup showed osteocalcin (OC) expression. Further details regardingmethodology and results are provided in Example 7.

FIG. 25 is a series of microscopy images of Alizarin Red stainedsections of osteocalcin expression at 6 weeks with hMSCs with andwithout GF. Only the BMP2 layer in the +GF group showed osteocalcin (OC)expression. Further details regarding methodology and results areprovided in Example 7.

FIG. 26A is an image showing engineered tendon-bone interface (Smith etal., Connective Tissue Research, 53(2): 95-105, (2012)). Further detailsregarding methodology and results are provided in Example 7.

FIG. 26B is an immunofluorescence image showing region specificexpression of OC, Col-I, Col-II, and AGC showing the spatiotemporaldelivery system for engineering tendon-bone interfaces with native likefibrocartilaginous gradient matrix. Further details regardingmethodology and results are provided in Example 7.

FIG. 27A is an image of a 3D printed ligament-bone scaffold. Furtherdetails regarding methodology and results are provided in Example 7.

FIG. 27B is a series of fluorescence images of ligament-bone scaffold at6 weeks. Dense collagenous tissue formed in the +GF groups.

FIG. 27C is a series of fluorescence images of ligament-bone scaffold at6 weeks. Dense collagenous tissue formed in the +GF groups.

FIG. 27D is a series of images and fluorescence images of ligament-bonescaffold at 6 weeks. OC was expressed in the bone and interface regionsof the +GF groups.

FIG. 28A is a series of images showing H&E and Picrosirius Red stainingof the scaffolds after 6 weeks in vitro culture of hMSCs showing tissueformation in the scaffolds for rotator cuff repair. Further detailsregarding methodology and results are provided in Example 7.

FIG. 28B is an image of a scaffold design for bone-tendon multi-tissueformation with defined interface. Further details regarding methodologyand results are provided in Example 7.

FIG. 28C is an Alizarin Red staining showing mineralized tissueformation in the bone region (having BMP2 μS). −GF group was fabricatedwith empty PLGA μS embedded in the PCL microstrands. Further detailsregarding methodology and results are provided in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery thatmicrospheres encapsulating an active agent (e.g., a growth factor) canbe mixed within a molten polymer (e.g., PCL) and dispensed (e.g.,through a needle), creating biocompatible scaffold of micro-sized growthfibers that can provide for tissue formation or regeneration. Suchbiocompatible scaffold can be of an anatomical dimension and shape. Abiocompatible scaffold described herein can have fully integratedmicrochannels, mechanical stability supporting initial loadbearing, orgrowth factor delivery (e.g., via microspheres) to guide regeneration by(e.g., endogenous) progenitor cells (e.g., stem cells).

It is believed that the present disclosure is the first to achievetissue formation by micro-precise spatiotemporal delivery of multiplegrowth factors in an integrated 3D printed scaffold in vitro and invivo. Furthermore, such materials and techniques can avoid use ofharmful organic solvent and still provide an integrated spatiotemporaldelivery with custom-designed scaffold.

Conventional scaffolds made from artificial materials do not havesufficiently high spatial and temporal resolution required for theformation of tissues with distinct boundaries or interfaces. This canmake the engineering of complexes with multiple types of tissue (acommon characteristic of many natural body's tissue systems) difficultor impossible.

The potential to regenerate multi-tissue complexes (e.g., within thecraniofacial system) using 3D-printed multi-phase scaffolds has profoundclinical applications. Previous approaches of incorporatingbiodegradable microspheres (μS) on the surface of scaffolds can belimited by the technical difficulties of achieving even distribution ofgrowth factors, having precise control of spatial distribution, andmaintaining the scaffold's original microstructure. Approaches describedherein can overcome such obstacles, providing for a 3D-printed scaffoldfor integrated regeneration of multi-tissue complexes in, e.g., acraniofacial system.

The present disclosure provides, inter alia, a 3D-printing method thatcan achieve spatial-temporal resolved delivery of growth hormones in abiocompatible scaffold in vitro and in vivo. For example,poly(lactic-co-glycolic acid) (PLGA) microspheres encapsulating anactive agent (e.g., a growth factor) can be mixed within a moltenpolymer (e.g., PCL) and dispensed through a needle, creating micro-sizedfibers containing the scaffold and growth fibers that can provide fortissue formation. Such three-dimensional scaffolds can be customdesigned through the 3D-printer software or constructed through thedeposition of these fibers. As described herein, the concentration ofgrowth factors can be controlled. This ability, in conjunction with asmall needle size (e.g., about 50 μm to about 400 μm), can provide ahigh spatial-temporal resolution to be achieved. By allowing for theprecise formation of multiple tissues with distinct characteristics,methods and compositions described herein can allow for ready-to-implantgrafts to guide regeneration of multi-tissue interfaces.

As shown in Example 1, methods, systems, and compositions describedherein were used to improve the spatiotemporal resolution of thedelivery of components of an artificial tissue so as to create multipletissues with correct tissue interfaces. In brief, CTGF and TGFβ3 growthfactors were encapsulated in microspheres (10-400 μm) ofpoly(lactic-co-glycolic acid) (PLGA). Other growth factors can be used,including but not limited to, CTGF, TGFβs, BMPs (e.g., bonemorphogenetic growth factor 2 (BMP2)), SDF, bFGF, IGF, GDF, PDGF, VEGF,or EGF or their isoforms. The microspheres were added to the moltenpolycarprolactone (PCL) and dispensed via a 3D-printer through astainless steel needle (diameter range of about 50 μm to about 400 μm),effectively creating microfibers containing both the scaffold and growthfactors useful for tissue growth. 3D designs can be made or constructedby performing a layer-by-layer deposition of these microfibers. Suchprocedures can provide a micro-precise spatiotemporal delivery ofmultiple growth factors. Furthermore, by modulating the encapsulationagent, the release rate of encapsulated agents (e.g., growth factors)can be controlled. As reported in Example 1, after four weeks of culturewith human mesenchymal stem cells, the PCL scaffolds with PLGAencapsulated CTGF and TGFβ3 growth factors successfully producedformation of correct multi-tissue interfaces.

The methods, materials, and resulting scaffold of Example 1 weredifferent than conventional stereolithography or fused filamentfabrication (FFF) 3D printing techniques. In conventionalstereolithography 3D printing techniques, materials must bephotopolymerizable and do not include commonly used materials for tissueengineering, such as PCL, PLLA, or PLGA. Also, conventionalstereolithography does not allow for incorporation of multiple growthfactors in a single unit of scaffolds. In conventional FFF, fibers aredeposited and fused to form a bulk structure. Deposited microfibers ofExample 1 followed specific patterns for each layer to form both outershape as well as internal microchannel structures. Such porosity can beimportant for tissue engineering scaffolds. Furthermore, Example 1demonstrates maintenance of a safe level of temperature in microspheresencapsulating an agent, which was confirmed by heat-diffusion analysisand stem cell culture experiment.

Multi tissue interfaces, which can be present in real tissueenvironments, can require precise spatial-temporal resolution of growthfactors. The present disclosure can overcome the lack of spatial ortemporal resolution of conventional design methods that may be unable toproduce biomaterials that accurately mimic the natural tissue. Methods,systems, and compositions described herein can use 3D-printing toincrease the spatiotemporal resolution of tissue design, in someembodiments using specific blends of polymers and growth factors withmicron-sized needles to construct a three-dimensional scaffold. Theincreased resolution of this delivery system can allow for design ofmulti-tissue complexes with well-defined interfaces. Such technology canprovide a powerful technique for the custom design of scaffolds withprecise control over the composition of tissue growth factors.

As shown herein, various biomaterial-based scaffolds combined withdelivery of growth factors have been tested to support regeneration ofconnective tissues and their interfaces. Previous scaffold designs,including stratified scaffolds with multiple phases or scaffolds withgradient growth factor/composition, have failed to recapitulate theintegrated and micro-scale interfaces between multiple tissues withdistinct characteristics, the pivotal features of native structures.Described herein are methods, systems, and compositions that can achievea micro-precise spatiotemporal delivery of a growth factor(s) embeddedin 3D-Printing that has potential to guide regeneration of integratedmulti-tissue interfaces.

Methods, systems, and compositions described herein can provide aspatiotemporal delivery system embedded in 3D-Printed scaffolds that canserve as a ready-to-implant grafts to guide regeneration of multi-tissuecomplexes or interfaces. For example, the spatiotemporal delivery systemcan include microspheres with an encapsulated growth factor. As anotherexample, multi-tissue complexes or interfaces can include, but are notlimited to the musculoskeletal system, craniofacial system,periodontium, cementum (CM)-periodontal ligament (PDL)-alveolar bone(AB) complex, ligament/tendon-to-bone insertion, rotator cuff,supraspinatus tendon-to-bone interface (e.g., between tendon,fibrocartilage, and bone), supraspinatus tendon-fibrocartilage(unmineralized and mineralized)-bone interface, articularcartilage-to-bone junction, anterior cruciate ligament (ACL)-to-bonecomplex, anterior cruciate ligament-fibrocartilage-bone interface,intervertebral disc (nucleus pulposus-annulus fibrosus-endplates),cementum-periodontal ligament-alveolar bone, muscle-to-tendon,inhomogeneous or anisotropic tissues such as knee meniscus ortemporomandibular joint (TMJ) disc, root-periodontium complex,tendon-bone insertion, synovial joints, or fibrocartilaginous tissues.

Methods, systems, or compositions described herein can be used fordesign of grafts for, inter alia, the regeneration of multi-tissueinterfaces such as those found in the musculoskeletal system; grafts forinhomogeneous and anisotropic tissues such as TMJ or knee meniscus;delivery of polymer embedded alloyed materials; or dispensing conductingpolymeric materials in a controlled manner.

Multi-Tissue Complex

As described herein, a biocompatible scaffold can form or regenerate amulti-tissue complex. A multi-tissue complex can include a multi-tissueinterface. A multi-tissue interface can have regional variance in celland matrix type, well-defined multi-phase tissues (e.g., ACL-to-bonecomplex), or inhomogeneous or anisotropic multi-phase tissues (e.g.,TMJ, knee meniscus tissues). For example, multi-tissue complexes orinterfaces can include, but are not limited to the musculoskeletalsystem; craniofacial system; periodontium, cementum (CM)-periodontalligament (PDL)-alveolar bone (AB) complex; ligament/tendon-to-boneinsertion; rotator cuff; supraspinatus tendon-to-bone interface (e.g.,between tendon, fibrocartilage, or bone); supraspinatustendon-fibrocartilage (unmineralized and mineralized)-bone interface;articular cartilage-to-bone junction; anterior cruciate ligament(ACL)-to-bone complex; anterior cruciate ligament-fibrocartilage-boneinterface; intervertebral disc (nucleus pulposus-annulusfibrosus-endplates); cementum-periodontal ligament-alveolar bone;muscle-to-tendon; inhomogeneous or anisotropic tissues such as kneemeniscus or temporomandibular joint (TMJ) disc; root-periodontiumcomplex; tendon-bone insertion; synovial joints; or fibrocartilaginoustissues.

Conventionally, it was thought to be a challenge to achieve functionalregeneration of multi-tissue complex with interfaces between distincttissues, including ligament/tendon-to-bone. It is generally believedthat almost all connective tissues function as an integrated unitcomprised of multiple tissues with distinct cell population ormechanical properties. Until now, no reliable tool was available forsuccessful regeneration of multi-tissue complex with integratedinterface. Because many connective tissues function as an integratedunit, comprising multiple tissues with distinct cell populations andbiochemical or mechanical properties, one of the challenges infunctional tissue regeneration addressed by the constructs and methodsdisclosed herein, establishes biochemically and physically integratedmulti-tissue complex or multi-tissue construct and inhomogeneous tissuewith a regional variance in cell and matrix types. Methods andconstructs, as described herein, can be used to regenerate suchmultiphase tissues.

As an example, a multiphase tissue can be a temporomandibular joint(TMJ) or knee meniscus. TMJ is a multiphase fibrocartilaginous tissuewith collagen-rich peripheral bands and fibrocartilage in intermediatezone. A TMJ disc is a heterogeneous fibrocartilaginous tissue positionedbetween mandibular condyle and glenoid fossa of the temporal bone, withimportant roles in TMJ functions. Tissue engineering a TMJ disc can bean approach to overcoming limitations of current treatments for TMJdisorders. Similarly, a knee meniscus shows a gradient change fromdensely aligned collagenous matrix in the outer zone to the avascularcartilaginous matrix in the inner zone.

The regenerated multi-tissue complex can have mechanical propertiessimilar to the corresponding native tissue complex. For example,mechanical properties of the regenerated tissue complex can includetensile modulus, compressive modulus, instantaneous modulus (Ei),relaxation modulus (Er), or coefficient of viscosity (μ). For example,the compressive modulus of a regenerated multi-tissue complex can bemore than about 1 MPa to about 60 MPa. As another example, the tensilemodulus can be at least about 10 MPa to about 70 MPa. As anotherexample, the instantaneous modulus (Ei) can be at least about 2 MPa toabout 25 MPa. As another example, the relaxation modulus (Er) can be atleast about 0.5 MPa to about 20 MPa. As another example, the ratio,Er/Ei can be at least about 0.1 MPa to about 1.5 MPa. As anotherexample, the coefficient of viscosity (μ) can be at least about 10 MPa·sto about 50 MPa·s. It is understood that recitation of the above rangesincludes discrete values between each recited range.

The regenerated multi-tissue complex can have properties similar to thecorresponding native tissue complex. For example, properties can includecartilage thickness, OARSI score, or GAG content. For example, thecollagen content can be at least about 2 mg per g of wet scaffold toabout 14 mg per g of wet scaffold. As another example, the GAG contentcan be at least about 0.5 μg per gram of wet scaffold to about 5 μg pergram of wet scaffold. As another example, the OARSI score can be lessthan 6. As another example, the collagen thickness can be at least about0.4 mm to about 1 mm. It is understood that recitation of the aboveranges includes discrete values between each recited range.

Scaffold

As described herein, a scaffold can be produced according to a threedimensional (3D) printing method such that threads or fibers of thescaffold include one or more microsphere encapsulated agents distributedthrough all or part of the scaffold. For example, 3D printing can referto various additive manufacturing processes for fabricating 3D objectsvia layer-by-layer deposition of materials.

For example, the scaffold can be an integrated 3D printed scaffold withmultiphase micro-architecture and regional distribution of a growthfactor or multiple growth factors that can provide efficientbiochemical/physical scaffolding environment for regeneration ofmultiphase tissues. For example, μS encapsulated with a growth factor orvarious growth factors can be incorporated in a 3D printing matrixmaterial prior to 3D printing a scaffold.

As another example, 3D printed scaffolds, as described herein, can becustom-designed with readily tunable microstructure and porosity, andavailable in a wide range of compatible materials.

The 3D printing technique, as described herein, enabled construction ofready-to-implant scaffolds with native-like microfiber orientation andspatiotemporal GFs delivery, subsequently leading to multi-tissueregeneration and improved healing. For example, the in vivo findings ofenhanced disc healing by 3D printed scaffolds can have a significantclinical impact in the treatment for ligament disorders, such as TMJdisorders (see e.g., Example 5).

Previous approaches provided controlled delivery of multiple growthfactors in different regions in a 3D printed scaffold, leading toregeneration of integrated multiphase tissues from a singlestem/progenitor cells, but incorporation of μS on the surface ofmicrostrands suffered from limitations, including: 1) the efficiency ofμS incorporation was highly dependent on microchannel structure, 2) theresolution of the manually controlled spatial μS distribution was low,and 3) the incorporated μS on the microstrands disruptedinterconnectivity, surface tomography, and micro-pattern in scaffolds.

It is currently believed that no previous approach has reconstructed theunique anatomical shape, the heterogeneous biochemicalcomposition/orientation, and the associated anisotropic mechanicalproperties of the multi-tissue interfaces as described herein (e.g., TMJdisc). Further, previous methods have been limited by low resolution inspatial control, a restricted range of applicable materials andmechanical properties, and lack spatiotemporal release control of morethan one bioactive cue (see e.g., Bose, 2013; 16:496-502).

The methods and compositions described herein overcome conventionalmethod limitations by the development of an advanced spatiotemporaldelivery system embedded in 3D printed scaffolds. By embeddingμS-encapsulating a growth factor or a plurality of growth factors in apolymeric microstrand constituting a 3D structure, micro-precisespatiotemporal delivery in anatomically correct 3D printed scaffolds wasachieved. As described herein, the novel 3D printed scaffold embeddedwith a micro-precise spatiotemporal delivery system can successfullyguide formation of multi-tissue interfaces from mesenchymalstem/progenitor cells (MSCs) in a resolution sufficient to recapitulatenative tissue interfaces. For example, TMJ disc scaffolds, spatiallyembedded with connective tissue growth factor (CTGF)- and TGFβ3-μS,significantly improved healing of perforated rabbit TMJ discs withmultiphase fibrocartilaginous tissues. It is noted that spatial controlof embedded active agent can include the presence of the embedded agentor the absence of the embedded region, e.g., in a region, layer, orother structure of the scaffold.

Various 3D printing methods are summarized in, for example, Sawkins etal. 2013 Recent Patents on Biomedical Engineering 6, 2-13; or Li et al.2014 International Journal of Polymer Science, Article ID 829145, 1-13.Except as otherwise noted herein, therefore, methods, systems, andcompositions described herein can be carried out in accordance with, oradapted to, such processes.

A scaffold can be fabricated with any matrix material recognized asuseful by the skilled artisan. Choice of scaffold material can becompatible with the 3D printing method employed. A matrix material canbe a biocompatible material that generally forms a porous, microcellularscaffold, which provides a physical support for cells migrating thereto.Such matrix materials can: allow cell attachment or migration; deliveror retain cells or biochemical factors; enable diffusion of cellnutrients or expressed products; or exert certain mechanical orbiological influences to modify the behavior of the cell phase. The 3Dprinted scaffold can be a porous, microcellular scaffold of abiocompatible material that provides a physical support or an adhesivesubstrate for recruitment or growth of cells during in vitro or in vivoculturing.

Suitable scaffold or matrix materials are discussed in, for example, Maand Elisseeff, ed. (2005) Scaffolding In Tissue Engineering, CRC, ISBN1574445219; Saltzman (2004) Tissue Engineering: Engineering Principlesfor the Design of Replacement Organs and Tissues, Oxford ISBN019514130X. For example, matrix materials can be, at least in part,solid xenogenic (e.g., hydroxyapatite) (Kuboki et al. 1995 ConnectTissue Res 32, 219-226; Murata et al. 1998 Int J Oral Maxillofac Surg27, 391-396), solid alloplastic (polyethylene polymers) materials (Saitoand Takaoka 2003 Biomaterials 24 2287-93; Isobe et al. 1999 J OralMaxillofac Surg 57, 695-8), or gels of autogenous (Sweeney et al. 1995.J Neurosurg 83, 710-715), allogenic (Bax et al. 1999 Calcif Tissue Int65, 83-89; Viljanen et al. 1997 Int J Oral Maxillofac Surg 26, 389-393),or alloplastic origin (Santos et al. 1998. J Biomed Mater Res 41,87-94), or combinations of the above (Alpaslan et al. 1996 Br J of OralMaxillofac Surg 34, 414-418).

Scaffold materials particularly suited to different 3D printing methodsare discussed in, for example, Sawkins et al. 2013 Recent Patents onBiomedical Engineering 6, 2-13; or Li et al. 2014 International Journalof Polymer Science, Article ID 829145, 1-13.

The matrix comprising the scaffold can have an adequate porosity and anadequate pore size so as to facilitate cell recruitment and diffusionthroughout the whole structure of both cells and nutrients. The matrixcan be biodegradable providing for absorption of the matrix by thesurrounding tissues, which can eliminate the necessity of a surgicalremoval. The rate at which degradation occurs can coincide as much aspossible with the rate of tissue or organ formation. Thus, while cellsare fabricating their own natural structure around themselves, thematrix can provide structural integrity and eventually break down,leaving the neotissue, newly formed tissue or organ which can assume themechanical load. The matrix can be an injectable matrix in someconfigurations. The matrix can be delivered to a tissue using minimallyinvasive endoscopic procedures.

The scaffold can comprise one or more layers, each with the same ordifferent matrix materials or the same or different microencapsulatedactive agents. For example, a scaffold can comprises at least twolayers, at least three layers, at least four layers, or more. Amulti-layer scaffold can contain one or more independently selectedmicrosphere encapsulated growth factors in each of the layers. Amulti-layer scaffold can contain one or more independently selectedmatrix materials in each of the layers. As another example, a scaffoldcan comprise a first layer comprising a first matrix material and asecond layer comprising a second matrix material. As another example, ascaffold can comprise a first layer comprising microspheresencapsulating a first independently selected active agent (e.g., agrowth factor) and a second layer comprising microspheres encapsulatinga second independently selected active agent (e.g., a second growthfactor). It is understand that multiple layers can include the same ordifference matrix materials or the same or different microencapsulatedactive agents. As another example, a scaffold can comprise a first layercomprising microspheres encapsulating CTGF and a second layer comprisingmicrospheres encapsulating TGFβ3.

The scaffold can comprise one or more regions, each with the same ordifferent matrix materials or the same or different microencapsulatedactive agents. For example, a scaffold can comprise at least tworegions, at least three regions, at least four regions, or more. Suchregions can be independent, adjacent, non-adjacent, or overlapping. Asanother example, a scaffold can comprise a first region comprising afirst matrix material and a second layer comprising a second matrixmaterial. As another example, a scaffold can comprise a first regioncomprising microspheres encapsulating a first independently selectedactive agent (e.g., a growth factor) and a second region comprisingmicrospheres encapsulating a second independently selected active agent(e.g., a second growth factor). As another example, a scaffold cancomprise a first region comprising microspheres encapsulating CTGF and asecond region comprising microspheres encapsulating TGFβ3. As describedabove, such regions can be partially overlapping or substantiallyoverlapping. The one or more regions can also have the same, different,or no microencapsulated active agent in the polymeric microfibers.

The scaffold can comprise a matrix material formed of synthetic polymerssuitable for the 3D printing process employed. Such synthetic polymersinclude, but are not limited to, polyurethanes, polyorthoesters,polyvinyl alcohol, polyamides, polycarbonates, polyvinyl pyrrolidone,marine adhesive proteins, cyanoacrylates, analogs, mixtures,combinations or derivatives of the above. Alternatively, the matrix canbe formed of naturally occurring biopolymers. Such naturally occurringbiopolymers include, but are not limited to, fibrin, fibrinogen,fibronectin, collagen, or other suitable biopolymers. Also, the matrixcan be formed from a mixture of naturally occurring biopolymers orsynthetic polymers.

The scaffold can include one or more matrix materials including, but notlimited to, a collagen gel, a polyvinyl alcohol sponge, apoly(D,L-lactide-co-glycolide) fiber matrix, a polyglactin fiber, acalcium alginate gel, a polyglycolic acid mesh, polyester (e.g.,poly-(L-lactic acid) or a polyanhydride), a polysaccharide (e.g.alginate), polyphosphazene, polyacrylate, or a polyethyleneoxide-polypropylene glycol block copolymer. Matrices can be producedfrom proteins (e.g., extracellular matrix proteins such as fibrin,collagen, fibronectin), polymers (e.g., polyvinylpyrrolidone), orhyaluronic acid. Synthetic polymers can also be used, includingbioerodible polymers (e.g., poly(lactide), poly(glycolic acid),poly(lactide-co-glycolide), poly(caprolactone), polycarbonates,polyamides, polyanhydrides, polyamino acids, polyortho esters,polyacetals, polycyanoacrylates), degradable polyurethanes, non-erodiblepolymers (e.g., polyacrylates, ethylene-vinyl acetate polymers or otheracyl substituted cellulose acetates and derivatives thereof),non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinylfluoride, poly(vinylimidazole), chlorosulphonated polyolefins,polyethylene oxide, polyvinyl alcohol, Teflon®, or nylon.

In some embodiments, the scaffold can be formed from a matrix materialcomprising polycaprolactone (PCL). After 3D printing, the resultingpolymeric microfiber can also comprises PCL.

The scaffold can further comprise any other agent or bioactive molecule,for example, an antibiotic or an additional growth factor. In someembodiments, the scaffold can be strengthened, through the addition of,e.g., human serum albumin (HSA), hydroxyethyl starch, dextran, orcombinations thereof. Suitable concentrations of these compounds for usein the compositions of the application are known to those of skill inthe art, or can be readily ascertained without undue experimentation.The concentration of a compound or a composition in the scaffold canvary with the nature of the compound or composition, its physiologicalrole, or desired therapeutic or diagnostic effect. Methods, systems andcompositions described herein provide for tailored concentrations ofagents throughout the scaffold. Such tailored concentration of an agentcan be a therapeutically effective amount. A therapeutically effectiveamount can be generally a sufficient concentration of therapeutic agentto display the desired effect without undue toxicity. In addition tomicroencapsulated agents embedded in scaffold threads or fibers asdescribed herein, other agents can be incorporated into the scaffold ormatrix material by any known method. For example, microspheres (e.g.,PLGA microspheres) encapsulated with GFs can be embedded intomicrofibers comprised of a matrix material (e.g., PCL microfibers) inthe 3D scaffold. In some embodiments, additional agents can be embeddedin a gel, e.g., a collagen gel incorporated into the pores of thescaffold or matrix material or applied as a coating over a portion, asubstantial portion, substantially all of, or all of the scaffold ormatrix material.

Alternatively, chemical modification methods can be used to covalentlylink a compound or a composition to a matrix material. The surfacefunctional groups of the matrix can be coupled with reactive functionalgroups of a compound or a composition to form covalent bonds usingcoupling agents well known in the art such as aldehyde compounds,carbodiimides, and the like. Additionally, a spacer molecule can be usedto gap the surface reactive groups and the reactive groups of thebiomolecules to allow more flexibility of such molecules on the surfaceof the matrix. Other similar methods of attaching biomolecules to theinterior or exterior of a matrix will be known to one of skill in theart.

Channels of the scaffold can be engineered to be of various diameters.As described herein, the present disclosure provides for precise controland placement of micron sized fibers or strands. For example, thechannels of the scaffold can have an inter-strand spacing diameterranging from micrometers to millimeters. Microchannels generally have anaverage diameter of about 0.1 μm to about 1,000 μm, e.g., about 50 μm toabout 500 μm (for example about 100 μm, 150 μm, about 200 μm, about 250μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500μm, or about 550 μm). One skilled in the art will understand that thedistribution of microchannel diameters can have any distributionincluding a normal distribution or a non-normal distribution. In someembodiments, microchannels are a naturally occurring feature of thematrix material(s). It is understood that recitation of the abovediscrete values includes ranges of values between each recited discretevalue. In other embodiments, microchannels are engineered to occur inthe matrix materials.

In some embodiments, the scaffold comprises pores. Pores can enhance therelease profile of agents incorporated in the scaffold. Pores can beproduced on the scaffold by hydrolysis (e.g., with NaOH). Pores can bean inherent property of a given matrix material.

In some embodiments, the scaffold does not comprise a transplantedmammalian cell, e.g., no cell is applied to the scaffold or any cellpresent in the scaffold migrated into the scaffold. A scaffold isgenerally understood to be a three-dimensional structure into whichcells, tissue, vessels, etc., can grow, colonize, or populate when thescaffold is placed into a tissue site. A scaffold of the method can beas discussed herein.

Fabrication

As shown herein, a matrix material (e.g., PCL) containing microsphere(e.g., PLGA microsphere) encapsulated active agent (e.g., a growthfactor such as CTGF, TGFβ3, or BMP) can be heated up to 100° C. whilekeeping about 80% of total volume of microspheres lower than about 45°C. (see e.g., Example 1; FIG. 2 ). Exemplary results showed that 10 mgto 100 mg of PLGA microspheres could be controllably embedded in 1 g ofPCL fibers. Ratios of PCL to PLGA μS can be optimized to yield a desired(e.g., an even) distribution of μS throughout the 3D-Printed scaffolds.

Various 3D printing methods are summarized in, for example, Sawkins etal. 2013 Recent Patents on Biomedical Engineering 6, 2-13; or Li et al.2014 International Journal of Polymer Science, Article ID 829145, 1-13.Except as otherwise noted herein, therefore, methods, systems, andcompositions described herein can be carried out in accordance with, oradapted to, such processes. Various devices for deposition of continuousmicrofibers are known (e.g., 3D Bioplotter® (EnvisionTec, Germany)) andcan be adapted for materials and processes described herein.

3D printing, also known as additive manufacturing (AM), can refer tovarious processes used to synthesize a three-dimensional object. Using a3D printing device, successive layers of material can be formed undercomputer control to create an object, such as a polymeric fiber orscaffold described herein. These objects can be of almost any shape orgeometry and can produced from a 3D model or other electronic datasource. A 3D printing device can include a computer having a memory forstoring and executing a digital file containing machine-interpretableinstructions for forming a 3D scaffold described herein. A 3D printingdevice can include one or more cartridges for containing a matrixmaterial or microencapsulated active agent to be dispensed (see e.g.,FIG. 1A). A 3D printing device can include a heater for heating a matrixmaterial or microencapsulated active agent to a temperature sufficientto be dispersed through the device. A 3D printing device can include aprinting needle (see e.g., FIG. 1A) to dispense, e.g., from one or morecartridges, a polymeric microfiber optionally having microencapsulatedactive agent distributed throughout. Guiding of the printing needle canbe according to a digital file containing machine-interpretableinstructions for a layer-by-layer deposition of continuous microfibersfrom the first cartridge through the printing needle so as to form apre-defined 3D scaffold structure. A 3D printing device can include agantry optimized for movement in horizontal X and Y directions, with aslow climb in the Z direction as the scaffold is printed. Alternatively,a 3D printing device can use polar coordinates with radial gantrymovement to print scaffolds with circular symmetry. A 3D printing devicecan include a chamber, which can be maintained at a pre-selectedtemperature (e.g., just below melting point of the matrix material) soas to provide for successful bonding of successive polymericmicrofibers.

The 3D printing process can be according to fused filament fabrication,which uses a continuous filament of extruded thermoplastic material,which in view of the present disclosure can be a matrix material. Thiscontinuous filament can be fed from a large coil or from a cartridgecontaining matrix material or microencapsulated active agents, through amoving, heated printer extruder head. In some embodiments, one or morecartridges each contain matrix material (e.g., pellets, chips, or othersmall particles). In some embodiments, one or more cartridges eachcontain matrix material (e.g., pellets, chips, or other small particles)and microsphere encapsulated active agents. In some embodiments, (i) oneor more cartridges each contain matrix material (e.g., pellets, chips,or other small particles) or (ii) one or more other cartridges eachcontain matrix material (e.g., pellets, chips, or other small particles)and microsphere encapsulated active agents. The cartridge can contain aheating element and the cartridge contents can be fluidically connectedto the extruding nozzle or needle. Molten material, at a temperature atwhich the matrix material is workable but the encapsulation materialdoes not exceed its melting point, can be forced out of a print head'snozzle or needle and can deposited on the growing scaffold. One or morecartridges can be switched during scaffold deposition to provide for,e.g., changes in matrix material, microencapsulated active agent(including different types or presence or absence), or for temperatureregulation (e.g., maintaining microencapsulated active agent at ourbelow a threshold temperature as described further herein). Thedeposition head (e.g., nozzle or needle) can be moved, under computercontrol, to define the printed scaffold shape. In some embodiments, thehead moves in layers, moving in two dimensions to deposit one horizontalplane at a time, before moving slightly upwards to begin a new slice.The speed of the extruder head can also be controlled, to stop and startdeposition and form an interrupted plane without stringing or dribblingbetween sections, regions, or layers.

The disclosure herein can provide for forming a polymeric fiber having amicroencapsulated agent distributed in the polymeric fiber. At least oneagent can be encapsulated in a plurality of microspheres. Themicroencapsulated agent can be combined with a matrix materialscompatible with a 3D printing device. The matrix material andmicroencapsulated agent can be (separately or as a preformedcombination) introduced into a first cartridge of a 3D printing device.The matrix material and microencapsulated agent can be heated to atemperature sufficient to allow dispensing of the combination ofmicrospheres and matrix material. This temperature will depend at leastin part on the choice of matrix material, the melting point of themicrosphere material, or the temperature sensitivity of the activeagent. The matrix material and microencapsulated agent can be heated toa temperature sufficient to allow dispensing of the combination ofmicrospheres and matrix material while preventing substantialdegradation of the microsphere or the agent encapsulated in themicrosphere. The heated combination of matrix material andmicroencapsulated active agent can be dispensed from the firstcartridge, e.g., through a printing needle, to form a fiber, e.g., apolymeric microfiber. Such a polymeric microfiber can havemicroencapsulated active agent distributed throughout by virtue of beingco-dispensed with the matrix material.

The disclosure herein can provide for forming a biocompatible scaffoldcomposed of polymeric fibers having a microencapsulated agentdistributed in the polymeric fiber. At least one agent can beencapsulated in a plurality of microspheres. The microencapsulated agentcan be combined with a matrix materials compatible with a 3D printingdevice. The matrix material and microencapsulated agent can be(separately or as a preformed combination) introduced into a firstcartridge of a 3D printing device. For example, matrix material pellets(e.g., PCL pellets) and growth factor-encapsulated microspheres can bemixed in a dispensing cartilages of a 3D printing device. The matrixmaterial and microencapsulated agent can be heated to a temperaturesufficient to allow dispensing of the combination of microspheres andmatrix material. This temperature will depend at least in part on thechoice of matrix material, the melting point of the microspherematerial, or the temperature sensitivity of the active agent. The matrixmaterial and microencapsulated agent can be heated to a temperaturesufficient to allow dispensing of the combination of microspheres andmatrix material while preventing substantial degradation of themicrosphere or the agent encapsulated in the microsphere. The heatedcombination of matrix material and microencapsulated active agent can bedispensed from the first cartridge, e.g., through a printing needle, toform a fiber, e.g., a polymeric microfiber. Such a polymeric microfibercan have microencapsulated active agent distributed throughout by virtueof being co-dispensed with the matrix material. A 3D scaffold can beformed by guiding the printing needle to dispense an array of polymericmicrofibers having microencapsulated active agent distributedthroughout. Guiding of the printing needle can be according to a digitalfile containing machine-interpretable instructions for a layer-by-layerdeposition of continuous microfibers from the first cartridge throughthe printing needle so as to form a pre-defined 3D scaffold structure.

A 3D printing device can include one or more cartridges for containing amatrix material, microencapsulated active agent, or various combinationsthereof. A 3D printing device can include one or more cartridges activeconcurrently or sequentially. A 3D printing device can include aplurality of cartridges active concurrently or sequentially. A 3Dprinting device can include a first plurality of cartridges and a secondplurality of cartridges active concurrently or sequentially. Forexample, formation of a 3D printed scaffold described herein can includea plurality of cartridges, each containing independently selected matrixmaterial, microencapsulated active agent, or various combinationsthereof. For example, formation of a 3D printed scaffold describedherein can include 1, 2, 3, 4, 5 6, 7, 8, 9, 10, or more cartridges,each containing independently selected matrix material,microencapsulated active agent, or various combinations thereof.Multiple cartridges can share or have independent elements such as aheating element or printing needle. For example, multiple cartridges caneach contain an independent heating element (e.g., having same ordifferent temperature or melting profiles) or printing needle (e.g.,having same or different inner diameter). A 3D printing device canswitch cartridges before, during or after polymeric microfiber formationor scaffold formation. For example, printing cartridges containing thesame or different matrix materials or microencapsulated active agents orprinting needle size can be switched during fabrication of a polymericmicrofibril or during fabrication of a layer, region, or other structureof the scaffold so as to provide, e.g., differing spatial compositionsof matrix and active agent. As another example, printing cartridgescontaining the same or different matrix materials or microencapsulatedactive agents or printing needle size can be switched to controltemperature of the microencapsulated active agents, e.g., to maintainthe microencapsulated active agents below a threshold temperature (asfurther described herein).

In some embodiments, the melting point of the encapsulation material ishigher than the melting point of the matrix material. For example, themelting point of PLGA is over 200° C., while matrix materials can beselected having lower melting temperatures (e.g., PCL Tm˜100° C.;poly(butylene succinate) (PBS) Tm˜90-120° C.; Poly(ethylene oxide) (PEO)Tm˜65° C.; Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) Tm˜108° C.).By ensuring the melting point of encapsulation material is higher (e.g.,substantially higher) than the melting point of a matrix material, suchmicrospheres can maintain their original structure during the dispensingprocess (see e.g., FIG. 1B).

In some embodiments, the matrix material is heated such that themajority of the total volume of microspheres does not exceed apre-determined threshold of temperature or time such that the activeagent is damaged or destroyed. Where an active agent includes a protein(e.g., a protein growth factor), such protein will generally denaturewhen exposed to temperatures over about 65° C. for longer than 30minutes. As shown herein, a PCL matrix material containing PLGAmicrosphere encapsulated active agent (e.g., a growth factor such asCTGF, TGFβ3, or BMP) could be heated up to 100° C. for 30 minutes whilekeeping about 80% of total volume of microspheres lower than about 45°C., thus protecting the encapsulated agent.

In some embodiments, the matrix material is heated to about its meltingtemperature for a time no longer than would result in the equivalent ofabout 80% of total volume of microspheres reaching 65° C. for more than30 minutes. FIG. 2 shows a temperature of % total volume of PLGAmicrospheres versus time at 100° C. PCL melting temperature. It isunderstood that the actual data of FIG. 2 is exemplary and therelationship of % total volume, threshold temperature of microspheres,and time can be adapted to other combinations of matrix materials andencapsulation materials.

For example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.9 such that 90% oftotal volume of microspheres do not reach more than 65° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.8such that 80% of total volume of microspheres do not reach more than 65°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.7 such that 70% of total volume of microspheres do not reachmore than 65° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.6 such that 60% of total volume ofmicrospheres do not reach more than 65° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.5 such that 50% oftotal volume of microspheres do not reach more than 65° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.4such that 40% of total volume of microspheres do not reach more than 65°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.3 such that 30% of total volume of microspheres do not reachmore than 65° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.2 such that 20% of total volume ofmicrospheres do not reach more than 65° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.1 such that 10% oftotal volume of microspheres do not reach more than 65° C. for more than30 minutes. It is understood that the matrix material can be heated forlonger periods of time where the melting temperature is lower. It isunderstood that the actual data of FIG. 2 is exemplary and therelationship of % total volume, threshold temperature of microspheres,and time can be adapted to other matrix materials and encapsulationmaterials.

For example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.9 such that 90% oftotal volume of microspheres do not reach more than 60° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.8such that 80% of total volume of microspheres do not reach more than 60°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.7 such that 70% of total volume of microspheres do not reachmore than 60° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.6 such that 60% of total volume ofmicrospheres do not reach more than 60° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.5 such that 50% oftotal volume of microspheres do not reach more than 60° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.4such that 40% of total volume of microspheres do not reach more than 60°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.3 such that 30% of total volume of microspheres do not reachmore than 60° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.2 such that 20% of total volume ofmicrospheres do not reach more than 60° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.1 such that 10% oftotal volume of microspheres do not reach more than 60° C. for more than30 minutes. It is understood that the matrix material can be heated forlonger periods of time where the melting temperature is lower.

For example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.9 such that 90% oftotal volume of microspheres do not reach more than 55° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.8such that 80% of total volume of microspheres do not reach more than 55°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.7 such that 70% of total volume of microspheres do not reachmore than 55° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.6 such that 60% of total volume ofmicrospheres do not reach more than 55° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.5 such that 50% oftotal volume of microspheres do not reach more than 55° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.4such that 40% of total volume of microspheres do not reach more than 55°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.3 such that 30% of total volume of microspheres do not reachmore than 55° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.2 such that 20% of total volume ofmicrospheres do not reach more than 55° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.1 such that 10% oftotal volume of microspheres do not reach more than 55° C. for more than30 minutes. It is understood that the matrix material can be heated forlonger periods of time where the melting temperature is lower.

For example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.9 such that 90% oftotal volume of microspheres do not reach more than 50° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.8such that 80% of total volume of microspheres do not reach more than 50°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.7 such that 70% of total volume of microspheres do not reachmore than 50° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.6 such that 60% of total volume ofmicrospheres do not reach more than 50° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.5 such that 50% oftotal volume of microspheres do not reach more than 50° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.4such that 40% of total volume of microspheres do not reach more than 50°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.3 such that 30% of total volume of microspheres do not reachmore than 50° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.2 such that 20% of total volume ofmicrospheres do not reach more than 50° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.1 such that 10% oftotal volume of microspheres do not reach more than 50° C. for more than30 minutes. It is understood that the matrix material can be heated forlonger periods of time where the melting temperature is lower.

For example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.9 such that 90% oftotal volume of microspheres do not reach more than 45° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.8such that 80% of total volume of microspheres do not reach more than 45°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.7 such that 70% of total volume of microspheres do not reachmore than 45° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.6 such that 60% of total volume ofmicrospheres do not reach more than 45° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.5 such that 50% oftotal volume of microspheres do not reach more than 45° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.4such that 40% of total volume of microspheres do not reach more than 45°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.3 such that 30% of total volume of microspheres do not reachmore than 45° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.2 such that 20% of total volume ofmicrospheres do not reach more than 45° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.1 such that 10% oftotal volume of microspheres do not reach more than 45° C. for more than30 minutes. It is understood that the matrix material can be heated forlonger periods of time where the melting temperature is lower.

For example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.9 such that 90% oftotal volume of microspheres do not reach more than 40° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.8such that 80% of total volume of microspheres do not reach more than 40°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.7 such that 70% of total volume of microspheres do not reachmore than 40° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.6 such that 60% of total volume ofmicrospheres do not reach more than 40° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.5 such that 50% oftotal volume of microspheres do not reach more than 40° C. for more than30 minutes. As another example, the matrix material can be heatedaccording to the temperature and time profile of FIG. 2 for the r/R=0.4such that 40% of total volume of microspheres do not reach more than 40°C. for more than 30 minutes. As another example, the matrix material canbe heated according to the temperature and time profile of FIG. 2 forthe r/R=0.3 such that 30% of total volume of microspheres do not reachmore than 40° C. for more than 30 minutes. As another example, thematrix material can be heated according to the temperature and timeprofile of FIG. 2 for the r/R=0.2 such that 20% of total volume ofmicrospheres do not reach more than 40° C. for more than 30 minutes. Asanother example, the matrix material can be heated according to thetemperature and time profile of FIG. 2 for the r/R=0.1 such that 10% oftotal volume of microspheres do not reach more than 40° C. for more than30 minutes. It is understood that the matrix material can be heated forlonger periods of time where the melting temperature is lower.

As another example, the matrix material can be heated to about itsmelting temperature such that 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or10% of the total volume of microspheres do not reach more than 60° C.for more than about 10-40 minutes (e.g., 30 minutes). As anotherexample, the matrix material can be heated to about its meltingtemperature such that 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% ofthe total volume of microspheres do not reach more than 55° C. for morethan about 10-40 minutes (e.g., 30 minutes). As another example, thematrix material can be heated to about its melting temperature such that90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the total volume ofmicrospheres do not reach more than 50° C. for more than about 10-40minutes (e.g., 30 minutes). As another example, the matrix material canbe heated to about its melting temperature such that 90%, 80%, 70%, 60%,50%, 40%, 30%, 20%, or 10% of the total volume of microspheres do notreach more than 45° C. for more than about 10-40 minutes (e.g., 30minutes). As another example, the matrix material can be heated to aboutits melting temperature such that 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, or 10% of the total volume of microspheres do not reach more than40° C. for more than about 10-40 minutes (e.g., 30 minutes). It isunderstood that recitation of the above discrete values (e.g., % totalvolume) includes ranges between each value. It is understood thatrecitation of the above ranges (e.g., time) includes discrete valuesbetween each range.

Dispensing temperature can be a function on the choice of matrixmaterial and microsphere material. In some embodiments, dispensingtemperature is about the melting temperature of the matrix material. Forexample, a matrix material including PCL can be dispensed at about100-110° C. As another example, a matrix material including PBS can bedispensed at about 90-120° C. As another example, a matrix materialincluding PEO can be dispensed at about 65-75° C. As another example, amatrix material including PHBV can be dispensed at about 105-110° C.These exemplified matrix material dispensing temperatures can be below,e.g., the 200° C. melting temperature of PLGA microspheres or the120-130° C. melting temperature of higher molecular weight polyethylenemicrospheres. It is understood that the present disclosure can beadapted according to the melting point of the chosen microspherematerial.

Dispensing time can be a function on the choice of matrix material andmicrosphere material. In general, the longer a matrix materialcontaining a microsphere encapsulated active agent is dispensed, thehigher the temperature the total volume of microspheres can reach.

In some embodiments, the matrix material containing a microsphereencapsulated active agent is heated and dispensed within about 1 minute,about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes,about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes,about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes,about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes,about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes,about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes,about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes,about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes,about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes,about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes,about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes,about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes,about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes,about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes,about 58 minutes, about 59 minutes, or about 60 minutes, or more. It isunderstood that recitation of the above discrete values includes rangesbetween each value. For example, the matrix material containing amicrosphere encapsulated active agent can be heated and dispensed withinabout 10 minutes to about 45 minutes. As another example, the matrixmaterial containing a microsphere encapsulated active agent can beheated and dispensed within about 15 minutes to about 40 minutes. Asanother example, the matrix material containing a microsphereencapsulated active agent can be heated and dispensed within about 20minutes to about 35 minutes. As another example, the matrix materialcontaining a microsphere encapsulated active agent can be heated anddispensed within about 30 minutes. Where relatively loner periods oftime are desired or necessary (e.g., greater than 30 minutes) to print ascaffold, multiple printing cartridges can be used so as to avoidexcessive heating of one batch combination of matrix material andmicroencapsulated active agent.

A printing needle of a 3D printing device can be any suitable material(e.g., stainless steel) have a defined inner diameter (e.g., 50 μm to400 μm). As described herein, inner diameter of the printing nozzle orneedle is generally larger than the mean diameter of a polymericmicrosphere encapsulating an active agent so as to allow unimpeded,relatively unimpeded, or substantially unimpeded flow of unmelted orundamaged microencapsulated active agent with the molten matrixmaterial. In some embodiments, the inner diameter of the printing needleis larger than the mean diameter of the microspheres (e.g., about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, about 100%, about 150%, about 200%, about 250%, about300%, about 350%, about 400%, about 450%, about 500%, or more. It isunderstood that recitation of the above discrete values includes rangesbetween each value).

The inner diameter of the needle can be selected so as to createmicro-sized growth factor/microsphere-embedded fibers of desiredthickness. For example, a printing needle can have an inner diameter ofabout 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160μm, about 170 μm, about 180 μm, about 190 μm, about 200 μm, about 210μm, about 220 μm, about 230 μm, about 240 μm, about 250 μm, about 260μm, about 270 μm, about 280 μm, about 290 μm, about 300 μm, about 310μm, about 320 μm, about 330 μm, about 340 μm, about 350 μm, about 360μm, about 370 μm, about 380 μm, about 390 μm, about 400 μm, about 410μm, about 420 μm, about 430 μm, about 440 μm, about 450 μm, about 460μm, about 470 μm, about 480 μm, about 490 μm, or about 500 μm. It isunderstood that recitation of the above discrete values includes rangesbetween each value.

Agent

As described herein, an active agent can be encapsulated in amicrosphere and incorporated into a scaffold thread or fiber via a 3Dprinting method.

The active agent can be a growth factor. The active agent can be agrowth factor selected from CTGF, TGFβs (e.g., TGFβ3), CTGF, BMPs, SDF,bFGF, IGF, GDF, PDGF, VEGF, EGF, or AM, Ang, autocrine motility factor,BDNF, EGF, EPO, FGFs, FBS, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF,KGF, MSF, GDF-8, NGF, PDGF, TPO, TCGF, TGF-α, TGF-β, TNF-α, VEGF, PGF,Interleukins (ILs), Renalase (RNLS) or their isoforms.

Agents, such as biologic drugs that can be added to compositions andmethods as described herein can include immunomodulators and otherbiological response modifiers. A biological response modifier generallyencompasses a biomolecule (e.g., peptide, peptide fragment,polysaccharide, lipid, antibody) that is involved in modifying abiological response, such as the immune response or tissue or organgrowth and repair, in a manner that enhances a particular desiredtherapeutic effect, for example, the cytolysis of bacterial cells or thegrowth of tissue- or organ-specific cells or vascularization. Biologicdrugs can also be incorporated directly into the matrix component. Thoseof skill in the art will know, or can readily ascertain, othersubstances which can act as suitable non-biologic and biologic drugs.

Compositions described herein can also be modified to incorporate anagent, such as a diagnostic agent, such as a radiopaque agent. Thepresence of such agents can allow the physician to monitor theprogression of wound healing occurring internally. Such compoundsinclude barium sulfate as well as various organic compounds containingiodine. Examples of these latter compounds include iocetamic acid,iodipamide, iodoxamate meglumine, iopanoic acid, as well as diatrizoatederivatives, such as diatrizoate sodium. Other contrast agents that canbe utilized in the compositions can be readily ascertained by those ofskill in the art and can include, for example, the use of radiolabeledfatty acids or analogs thereof.

The concentration of an agent in the composition can vary with thenature of the compound, its physiological role, or desired therapeuticor diagnostic effect. A therapeutically effective amount can begenerally a sufficient concentration of therapeutic agent to display thedesired effect without undue toxicity. A diagnostically effective amountcan be generally a concentration of diagnostic agent which can beeffective in allowing the monitoring of the integration of the tissuegraft, while minimizing potential toxicity. In any event, the desiredconcentration in a particular instance for a particular compound can bereadily ascertainable by one of skill in the art.

Encapsulation

As described herein, an active agent can be encapsulated in amicrosphere (μS) and incorporated into a scaffold thread or fiber via a3D printing method. Such microspheres are useful as slow releasecompositions. For example, growth factors can be micro-encapsulated toprovide for enhanced stability or prolonged delivery. Encapsulationvehicles include, but are not limited to, microparticles, liposomes,microspheres, or the like, or a combination of any of the above toprovide the desired release profile in varying proportions. Othermethods of controlled-release delivery of agents will be known to theskilled artisan. Moreover, these and other systems can be combined ormodified to optimize the integration/release of agents with the scaffoldthread or fiber according to the 3D printing method. For example, theagents encapsulated in the microspheres can have a total accumulatedrelease rate of from about 0% to about 50% over the course of at least42 days. It is understood that recitation of the above range includesdiscrete values between the recited range. One skilled in the art willunderstand that the distribution of release rate can have anydistribution including a normal distribution or a non-normaldistribution.

For example, the polymeric delivery system can be a polymericmicrosphere, e.g., a PLGA polymeric microspheres. A variety of polymericdelivery systems, as well as methods for encapsulating a molecule suchas a growth factor, are known to the art (see e.g., Varde and Pack 2004Expert Opin Biol Ther 4, 35-51). Polymeric microspheres can be producedusing naturally occurring or synthetic polymers and are particulatesystems in the size range of 0.1 to 500 μm. Polymeric microspheres canhave a mean diameter of about 0.1 μm, 0.5 μm, 10 μm, about 20 μm, about30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm,about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm,about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm,about 190 μm, about 200 μm, about 210 μm, about 220 μm, about 230 μm,about 240 μm, about 250 μm, about 260 μm, about 270 μm, about 280 μm,about 290 μm, about 300 μm, about 310 μm, about 320 μm, about 330 μm,about 340 μm, about 350 μm, about 360 μm, about 370 μm, about 380 μm,about 390 μm, about 400 μm, about 410 μm, about 420 μm, about 430 μm,about 440 μm, about 450 μm, about 460 μm, about 470 μm, about 480 μm,about 490 μm, or about 500 μm, or more. As described herein the meandiameter of a polymeric microsphere encapsulating an active agent isgenerally smaller than the inner diameter of the printing nozzle orneedle so as to allow unimpeded, relatively unimpeded, or substantiallyunimpeded flow of unmelted or undamaged microencapsulated active agentwith the molten matrix material.

Polymeric micelles and polymeromes are polymeric delivery vehicles withsimilar characteristics to microspheres and can also facilitateencapsulation and matrix integration of the compounds described herein.Fabrication, encapsulation, and stabilization of microspheres for avariety of payloads are within the skill of the art (see e.g., Varde &Pack (2004) Expert Opin. Biol. 4(1) 35-51). The release rate of themicrospheres can be tailored by type of polymer, polymer molecularweight, copolymer composition, excipients added to the microsphereformulation, and microsphere size. Polymer materials useful for formingmicrospheres include PLA, PLGA, PLGA coated with DPPC, DPPC, DSPC, EVAc,gelatin, albumin, chitosan, dextran, DL-PLG, SDLMs, PEG (e.g., ProMaxx),sodium hyaluronate, diketopiperazine derivatives (e.g., Technosphere),calcium phosphate-PEG particles, and/or oligosaccharide derivative DPPG(e.g., Solidose). Encapsulation can be accomplished, for example, usinga water/oil single emulsion method, a water-oil-water double emulsionmethod, or lyophilization. Several commercial encapsulation technologiesare available (e.g., ProLease®, Alkerme). Selection of an encapsulationagent can depend on the 3D printing method selected.

Progenitor Cells

As described herein, progenitor cells can be cultured with a scaffolddescribed herein so as to form tissues comprising correct multi-tissueinterfaces. Also as described, a scaffold (e.g., cellular scaffold oracellular scaffold) can be implanted in a subject so as to inducerecruitment or migration of a progenitor cell.

Various compositions and methods described herein provide forimplantation of a progenitor cell, recruitment of a progenitor cell, orinducing migration of a progenitor cell. A progenitor cell can be a cellthat is undifferentiated or partially undifferentiated, and can divideand proliferate to produce undifferentiated or partiallyundifferentiated cells or can differentiate to produce at least onedifferentiated or specialized cell. A progenitor cell can be apluripotent cell, which means that the cell can be capable ofself-renewal and of trans-differentiation into multiple tissue typesupon differentiation. Pluripotent progenitor cells include stem cells,such as embryonic stem cells and adult stem cells. A progenitor cell canbe a multipotent cell. A progenitor cell can be self-renewing. Forexample, a progenitor cell can be a stem cell. As another example, aprogenitor cell can be an adult stem cell. As another example, aprogenitor cell can be a mesenchymal stem cell. As another example, aprogenitor cell can be a human mesenchymal stem cell. As anotherexample, a progenitor cell can be a bone marrow derived mesenchymal stemcell.

Progenitor cells can be isolated, purified, or cultured by a variety ofmeans known to the art Methods for the isolation and culture ofprogenitor cells are discussed in, for example, Vunjak-Novakovic andFreshney (2006) Culture of Cells for Tissue Engineering, Wiley-Liss,ISBN-10 0471629359. For example, mesenchymal stem cells can be isolatedfrom bone marrow and culture-expanded as described in U.S. patentapplication Ser. No. 13/877,260, published as U.S. Pat Pub No.2014-0079739, incorporated herein by reference.

A progenitor cell can be comprised of, or derived from, an animal,including, but not limited to, mammals, reptiles, avians, horses, cows,dogs, cats, sheep, pigs, and chickens, or human.

Formulation

As described herein, an agent can be encapsulated into a microsphere andincluded in a scaffold thread or fiber via 3D printing. Such agent canbe a formulated agent. Also described herein, a scaffold of the presentdisclosure can be implanted in a subject. Such scaffold can includevarious pharmaceutically acceptable carriers or excipients.

The agents and compositions described herein can be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers or excipients as described in, for example, Remington'sPharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN:0781746736 (2005), incorporated herein by reference in its entirety.Such formulations will contain a therapeutically effective amount of abiologically active agent described herein, which can be in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the subject.

The term “formulation” refers to preparing a drug in a form suitable foradministration to a subject, such as a human. Thus, a “formulation” caninclude pharmaceutically acceptable excipients, including diluents orcarriers.

The term “pharmaceutically acceptable” as used herein can describesubstances or components that do not cause unacceptable losses ofpharmacological activity or unacceptable adverse side effects. Examplesof pharmaceutically acceptable ingredients can be those havingmonographs in United States Pharmacopeia (USP 29) and National Formulary(NF 24), United States Pharmacopeial Convention, Inc, Rockville, Md.,2005 (“USP/NF”), or a more recent edition, and the components listed inthe continuously updated Inactive Ingredient Search online database ofthe FDA. Other useful components that are not described in the USP/NF,etc. may also be used.

The term “pharmaceutically acceptable excipient,” as used herein, caninclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic, or absorption delaying agents. The useof such media and agents for pharmaceutical active substances is wellknown in the art (see generally Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofaras any conventional media or agent can be incompatible with an activeingredient, its use in the therapeutic compositions is contemplated.Supplementary active ingredients can also be incorporated into thecompositions.

A “stable” formulation or composition can refer to a composition havingsufficient stability to allow storage at a convenient temperature, suchas between about 0° C. and about 60° C., for a commercially reasonableperiod of time, such as at least about one day, at least about one week,at least about one month, at least about three months, at least aboutsix months, at least about one year, or at least about two years.

The formulation should suit the mode of administration. The agents ofuse with the current disclosure can be formulated by known methods foradministration to a subject using several routes which include, but arenot limited to, parenteral, pulmonary, oral, topical, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, buccal, and rectal. The individual agents may alsobe administered in combination with one or more additional agents ortogether with other biologically active or biologically inert agents.Such biologically active or inert agents may be in fluid or mechanicalcommunication with the agent(s) or attached to the agent(s) by ionic,covalent, Van der Waals, hydrophobic, hydrophilic or other physicalforces.

Controlled-release (or sustained-release) preparations may be formulatedto extend the activity of the agent(s) and reduce dosage frequency.Controlled-release preparations can also be used to effect the time ofonset of action or other characteristics, such as blood levels of theagent, and consequently affect the occurrence of side effects.Controlled-release preparations may be designed to initially release anamount of an agent(s) that produces the desired therapeutic effect, andgradually and continually release other amounts of the agent to maintainthe level of therapeutic effect over an extended period of time. Inorder to maintain a near-constant level of an agent in the body, theagent can be released from the dosage form at a rate that will replacethe amount of agent being metabolized or excreted from the body. Thecontrolled-release of an agent may be stimulated by various inducers,e.g., change in pH, change in temperature, enzymes, water, or otherphysiological conditions or molecules.

Agents or compositions described herein can also be used in combinationwith other therapeutic modalities, as described further below. Thus, inaddition to the therapies described herein, one may also provide to thesubject other therapies known to be efficacious for treatment of thedisease, disorder, or condition.

Therapeutic Methods

Also provided is a process of treating a tissue defect in a subject inneed of administration of a therapeutically effective amount of scaffolddescribed herein, so as to heal, repair, improve, or prevent a tissuedefect.

Treatment of a tissue defect can include regeneration, restoration, orformation of a multi-tissue complex comprising a tissue defect (e.g.,musculoskeletal injury, disease, disorder, or condition). For example,treatment of a tissue defect can include functional tendon restorationby regenerating integrated tendon-bone fibrocartilaginous interfaces.

A tissue defect can comprise or be associated with a multi-tissuecomplex or interface. For example, a multi-tissue complex or interfacecan include, but is not limited to the musculoskeletal system,craniofacial system, periodontium, cementum (CM)-periodontal ligament(PDL)-alveolar bone (AB) complex, ligament/tendon-to-bone insertion,rotator cuff, supraspinatus tendon-to-bone interface (e.g., betweentendon, fibrocartilage, and bone), supraspinatus tendon-fibrocartilage(unmineralized and mineralized)-bone interface, articularcartilage-to-bone junction, anterior cruciate ligament (ACL)-to-bonecomplex, anterior cruciate ligament-fibrocartilage-bone interface,intervertebral disc (nucleus pulposus-annulus fibrosus-endplates),cementum-periodontal ligament-alveolar bone, muscle-to-tendon,inhomogeneous or anisotropic tissues such as knee meniscus ortemporomandibular joint (TMJ) disc, root-periodontium complex,tendon-bone insertion, synovial joints, or fibrocartilaginous tissues.For example, musculoskeletal injuries can be associated with tendonsand/or ligaments.

As described herein, a biocompatible scaffold can overcome conventionalbarriers for meniscus regeneration, such as lack of fibrochondrocytes orcomplex bio/chemical structure and mechanics.

As an example, a subject in need can have a tissue defect of at leastabout 5%, about 10%, about 25%, about 50%, about 75%, about 90% or more,and compositions and methods described herein can provide an increase innumber or function of tissue. As another example, a subject in need canhave damage to a tissue or organ, and the method can provide an increasein biological function of the tissue or organ by at least about 5%,about 10%, about 25%, about 50%, about 75%, about 90%, about 100%, orabout 200%, or even by as much as about 300%, about 400%, or about 500%.The above discrete listings of values is understood to include rangesbetween each of the listed values. As yet another example, the subjectin need can have disease, disorder, or condition listed above, and themethod provides an engineered scaffold sufficient to recruit progenitorcells and form tissue with multi-tissue interfaces sufficient toameliorate or stabilize the disease, disorder, or condition. Forexample, the subject can have a disease, disorder, or condition thatresults in the loss, atrophy, dysfunction, or death of tissue cells. Ina further example, the subject in need can have an increased risk ofdeveloping a disease, disorder, or condition that can be delayed orprevented by the method. As yet another example, the subject in need canhave experienced death or dysfunction of tissue cells as the result of aside effect of a medication used for the treatment of another disease ordisorder.

Methods described herein are generally performed on a subject in needthereof. A subject in need of the therapeutic methods described hereincan be a subject having, diagnosed with, suspected of having, or at riskfor developing tissue defect. A determination of the need for treatmentwill typically be assessed by a history and physical exam consistentwith the disease or condition at issue. Diagnosis of the variousconditions treatable by the methods described herein is within the skillof the art. The subject can be an animal subject, including a mammal,such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys,hamsters, guinea pigs, and chickens, and humans. For example, thesubject can be a human subject. A subject can be an individual subject.A subject can be one or more subjects. A subject can be a plurality ofsubjects. A subject can be a subject population.

Implantation of an engineered construct is within the skill of the art.The scaffold can be either fully or partially implanted into a tissue ororgan of the subject to become a functioning part thereof. The implantcan initially attach to and communicate with the host through a cellularmonolayer. Over time, endogenous cells can migrate into the scaffold toform tissue. The cells surrounding the engineered tissue can beattracted by biologically active materials, including biologicalresponse modifiers, such as polysaccharides, proteins, peptides, genes,antigens, or antibodies, which can be selectively incorporated into thematrix to provide the needed selectivity, for example, to tether thecell receptors to the matrix, stimulate cell migration into the matrix,or both. The scaffold or matrix material can comprise a gelled phase andinterconnecting channels that allow for cell migration, augmented byboth biological and physical-chemical gradients. For example, cellssurrounding the implanted matrix can be attracted by biologically activematerials including CTGF, BMP, or TGFβ3. One of skill in the art willrecognize and know how to use other biologically active materials thatare appropriate for attracting cells to the matrix.

Generally, a safe and effective amount of a scaffold of the presentdisclosure is, for example, that amount that would cause the desiredtherapeutic effect in a subject while minimizing undesired side effects.In various embodiments, an effective amount of a scaffold of the presentdisclosure described herein can substantially heal, repair, improve, orprevent a tissue defect.

According to the methods described herein, administration can beparenteral, pulmonary, oral, topical, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,ophthalmic, buccal, or rectal administration.

When used in the treatments described herein, a therapeuticallyeffective amount of a scaffold of the present disclosure, or componentsthereof (e.g., active agents) can be employed in pure form or, wheresuch forms exist, in pharmaceutically acceptable salt form and with orwithout a pharmaceutically acceptable excipient. For example, thecompounds of the present disclosure can be administered, at a reasonablebenefit/risk ratio applicable to any medical treatment, in a sufficientamount to heal, repair, improve, or prevent a tissue defect.

The amount of a composition described herein that can be combined with apharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art thatthe unit content of agent contained in an individual dose of each dosageform need not in itself constitute a therapeutically effective amount,as the necessary therapeutically effective amount could be reached byadministration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectscan be the therapeutic index that can be expressed as the ratioLD₅₀/ED₅₀, where larger therapeutic indices are generally understood inthe art to be optimal.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the subject; the time ofadministration; the route of administration; the rate of excretion ofthe composition employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts (see e.g., Koda-Kimble etal. (2004) Applied Therapeutics: The Clinical Use of Drugs, LippincottWilliams & Wilkins, ISBN 0781748453; Winter (2003) Basic ClinicalPharmacokinetics, 4^(th) ed., Lippincott Williams & Wilkins, ISBN0781741475; Shamel (2004) Applied Biopharmaceutics & Pharmacokinetics,McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is wellwithin the skill of the art to start doses of the composition at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect can beachieved. If desired, the effective daily dose may be divided intomultiple doses for purposes of administration. Consequently, single dosecompositions may contain such amounts or submultiples thereof to make upthe daily dose. It will be understood, however, that the total dailyusage of the compounds and compositions of the present disclosure willbe decided by an attending physician within the scope of sound medicaljudgment.

Again, each of the states, diseases, disorders, and conditions,described herein, as well as others, can benefit from compositions andmethods described herein. Generally, treating a state, disease,disorder, or condition includes preventing or delaying the appearance ofclinical symptoms in a mammal that may be afflicted with or predisposedto the state, disease, disorder, or condition but does not yetexperience or display clinical or subclinical symptoms thereof. Treatingcan also include inhibiting the state, disease, disorder, or condition,e.g., arresting or reducing the development of the disease or at leastone clinical or subclinical symptom thereof. Furthermore, treating caninclude relieving the disease, e.g., causing regression of the state,disease, disorder, or condition or at least one of its clinical orsubclinical symptoms. A benefit to a subject to be treated can be eitherstatistically significant or at least perceptible to the subject or to aphysician.

Administration of a scaffold can occur as a single event or over a timecourse of treatment. For example, a scaffold can be administered daily,weekly, bi-weekly, or monthly. For treatment of acute conditions, thetime course of treatment will usually be at least several days. Certainconditions could extend treatment from several days to several weeks.For example, treatment could extend over one week, two weeks, or threeweeks. For more chronic conditions, treatment could extend from severalweeks to several months or even a year or more.

Treatment in accord with the methods described herein can be performedprior to, concurrent with, or after conventional treatment modalitiesfor a tissue defect.

A scaffold can be administered simultaneously or sequentially withanother agent, such as an antibiotic, an anti-inflammatory, or anotheragent. For example, a scaffold can be administered simultaneously withanother agent, such as an antibiotic or an anti-inflammatory.Simultaneous administration can occur through administration of separatecompositions, each containing one or more of a scaffold, an antibiotic,an anti-inflammatory, or another agent. Simultaneous administration canoccur through administration of one composition containing two or moreof a scaffold, an antibiotic, an anti-inflammatory, or another agent. Ascaffold can be administered sequentially with an antibiotic, ananti-inflammatory, or another agent. For example, a scaffold can beadministered before or after administration of an antibiotic, ananti-inflammatory, or another agent.

Administration

Agents and compositions described herein can be administered accordingto methods described herein in a variety of means known to the art. Theagents and composition can be used therapeutically either as exogenousmaterials or as endogenous materials. Exogenous agents are thoseproduced or manufactured outside of the body and administered to thebody. Endogenous agents are those produced or manufactured inside thebody by some type of device (biologic or other) for delivery within orto other organs in the body.

As discussed above, administration can be parenteral, pulmonary, oral,topical, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectaladministration.

Agents and compositions described herein can be administered in avariety of methods well known in the arts. Administration can include,for example, methods involving oral ingestion, direct injection (e.g.,systemic or stereotactic), implantation of cells engineered to secretethe factor of interest, drug-releasing biomaterials, polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, implantable matrix devices, mini-osmotic pumps,implantable pumps, injectable gels and hydrogels, liposomes, micelles(e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres(e.g., 1-100 μm), reservoir devices, a combination of any of the above,or other suitable delivery vehicles to provide the desired releaseprofile in varying proportions. Other methods of controlled-releasedelivery of agents or compositions will be known to the skilled artisanand are within the scope of the present disclosure.

Delivery systems may include, for example, an infusion pump which may beused to administer the agent or composition in a manner similar to thatused for delivering insulin or chemotherapy to specific organs ortumors. Typically, using such a system, an agent or composition can beadministered in combination with a biodegradable, biocompatiblepolymeric implant that releases the agent over a controlled period oftime at a selected site. Examples of polymeric materials includepolyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid,polyethylene vinyl acetate, and copolymers and combinations thereof. Inaddition, a controlled release system can be placed in proximity of atherapeutic target, thus requiring only a fraction of a systemic dosage.

Agents can be encapsulated and administered in a variety of carrierdelivery systems. Examples of carrier delivery systems includemicrospheres, hydrogels, polymeric implants, smart polymeric carriers,and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006)Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-basedsystems for molecular or biomolecular agent delivery can: provide forintracellular delivery; tailor biomolecule/agent release rates; increasethe proportion of biomolecule that reaches its site of action; improvethe transport of the drug to its site of action; allow colocalizeddeposition with other agents or excipients; improve the stability of theagent in vivo; prolong the residence time of the agent at its site ofaction by reducing clearance; decrease the nonspecific delivery of theagent to non-target tissues; decrease irritation caused by the agent;decrease toxicity due to high initial doses of the agent; alter theimmunogenicity of the agent; decrease dosage frequency, improve taste ofthe product; or improve shelf life of the product.

KITS

Also provided are kits. Such kits can include an agent or compositiondescribed herein and, in certain embodiments, instructions foradministration. Such kits can facilitate performance of the methodsdescribed herein. When supplied as a kit, the different components ofthe composition can be packaged in separate containers and admixedimmediately before use. Components include, but are not limited to ascaffold as described herein. Such packaging of the componentsseparately can, if desired, be presented in a pack or dispenser devicewhich may contain one or more unit dosage forms containing thecomposition. The pack may, for example, comprise metal or plastic foilsuch as a blister pack. Such packaging of the components separately canalso, in certain instances, permit long-term storage without losingactivity of the components.

Kits may also include reagents in separate containers such as, forexample, sterile water or saline to be added to a lyophilized activecomponent packaged separately. For example, sealed glass ampules maycontain a lyophilized component and in a separate ampule, sterile water,sterile saline or sterile each of which has been packaged under aneutral non-reacting gas, such as nitrogen. Ampules may consist of anysuitable material, such as glass, organic polymers, such aspolycarbonate, polystyrene, ceramic, metal or any other materialtypically employed to hold reagents. Other examples of suitablecontainers include bottles that may be fabricated from similarsubstances as ampules, and envelopes that may consist of foil-linedinteriors, such as aluminum or an alloy. Other containers include testtubes, vials, flasks, bottles, syringes, and the like. Containers mayhave a sterile access port, such as a bottle having a stopper that canbe pierced by a hypodermic injection needle. Other containers may havetwo compartments that are separated by a readily removable membrane thatupon removal permits the components to mix. Removable membranes may beglass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructionalmaterials. Instructions may be printed on paper or other substrate,and/or may be supplied as an electronic-readable medium, such as afloppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, and the like. Detailed instructions may not be physicallyassociated with the kit; instead, a user may be directed to an Internetweb site specified by the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biologyprotocols can be according to a variety of standard techniques known tothe art (see, e.g., Sambrook and Russel (2006) Condensed Protocols fromMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols inMolecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929;Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Green andSambrook 2012 Molecular Cloning: A Laboratory Manual, 4th ed., ColdSpring Harbor Laboratory Press, ISBN-10: 1605500569; Elhai, J. and Wolk,C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) ProteinExpr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production ofRecombinant Proteins: Novel Microbial and Eukaryotic Expression Systems,Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein ExpressionTechnologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein can be merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

In some embodiments the term “substantially” used in the context ofdescribing a particular embodiment (especially in the context of certainof the following claims) can be construed as “being largely but notwholly that which is specified”. Further, substantially can used toindicate that a value includes the standard deviation of the mean forthe device or method being employed to determine the value.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus can be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments that are disclosedand still obtain a like or similar result without departing from thespirit and scope of the present disclosure.

Example 1: 3D Printed Biocompatible Scaffold for Multi-Tissue Interface

The following example provides for a 3D printed biocompatible scaffoldwith spatiotemporal delivery of microsphere encapsulated CTGF and TGFβ3.

Selected growth factors (GF) were encapsulated inpoly(lactic-co-glycolic acids) (PLGA) microspheres (μS) for controlledrelease. Poly(lactic-co-glycolic acid) (PLGA) microspheres (10-400 μm)encapsulating CTGF, TGFβ3, and BMP growth factors, were prepared bydouble-emulsion technique.

Then 3D scaffolds with custom-designed microstructure and outer shapewere constructed using layer-by-layer deposition of continuous PCLmicrofibers. PCL pellets and growth factor-encapsulated microsphereswere mixed in dispensing cartilages of 3D Bioplotter® (EnvisionTec,Germany) and heated up to 100° C., selected from heating diffusionanalysis (see e.g., FIG. 1A). Material was dispensed through a finestainless steel needle (inner diameter 50 μm to 400 μm), creatingmicro-sized growth factor/microsphere-embedded PCL fibers.

Given the PLGA's melting point over 200° C., microspheres were able tomaintain their original structure during the dispensing process, asprotecting encapsulated GFs. Fluorescent dextran/μS was used to confirmthat PLGA microspheres were successfully embedded with an evendistribution in the 3D-deposited PCL microstrands (see e.g., FIG. 1B).The above approach provided for fabrication of a 3D scaffolds withmultiple growth factors delivered at desired location by changingdispending cartilage during the printing process, leading to fullyintegrated 3D custom-designed scaffold.

Results successfully demonstrated that 10 mg to 100 mg of PLGAmicrospheres were controllably embedded in 1 g of PCL fibers. Ratios ofPCL to PLGA μS can be optimized to yield a desired (e.g., an even)distribution of μS throughout the 3D-Printed scaffolds.

Results also showed that encapsulated growth factor inmicrosphere-embedded PCL scaffold had sustained release over 42 daysincubation in vitro (see e.g., FIG. 1E). Because the release rate can beadjusted by using different composition of PLGA, this approach enablesspatiotemporal delivery of multiple growth factors.

Temperature distribution within PLGA microspheres was determined uponheating surrounding PCL from 25° C. to 100° C. The heat conductiongoverning equation (eq. 1) was used for the calculation. Temperature asa function of r (radial position) and t (time) is plotted from 0 to 360minutes at 10 different depths (surface to core), where R is the radiusof microspheres. As shown in FIG. 2 , temperature inside themicrospheres was maintained lower than 45° C. during the fabricationprocess when heating the PCL to 100° C. Given the average fabricationtime for a scaffold (−30 mins), temperature over 80% of total volume ofmicrospheres is lower than 45° C., which can preserve bioactivity ofencapsulated growth factors. Such results are due at least in part tothe extremely low heat conductivity in PLGA and its high melting point.

Moreover, bioactivity of the loaded growth factors was confirmed by stemcell differentiation as shown in FIG. 3 .

The 3D printing process of this example was different than conventionalfused filament fabrication (FFF) techniques. In conventional FFF, fibersare deposited and fused to form a bulk structure whereas depositedmicrofibers of the present Example followed specific patterns for eachlayer to form both outer shape as well as internal microchannelstructures. Such porosity can be important for tissue engineeringscaffolds.

This procedure can provide for: (a) micro-precise spatiotemporaldelivery of one or more growth factors in selected locations in the3D-Printed scaffolds; (b) controlled release rate of each growth factorby changing compositions of PLGA; or (c) region-specific microstructuresin an integrated custom-designed scaffold.

Example 2: Growth Factor Delivery in 3D Printed Multi-Tissue InterfaceScaffold

The following Example demonstrates formation of multi-tissue interfacesin 3D-printed scaffold with spatiotemporal delivered CTGF and TGFβ3.Methods are according to Example 1 except as indicated otherwise.

A single layered rectangular structure was constructed (see e.g., FIG.3A) with alternative PCL microfibers-embedding CTGF-μS and TGFβ3-pS (seee.g., FIG. 3B). The size of microfibers and interfibers space was 100μm.

Then human bone marrow derived mesenchymal stem/progenitor cells (MSCs)were delivered in the scaffold's inter-fiber space via fibrin gel. After4 weeks culture in vitro, alternative depositions of COL-I+ and COL-II+matrices, corresponding to the pattern of growth factor delivery,forming an integrated interface within the 100— 200 μm interfiber zones(see e.g., FIG. 3C).

This demonstrated PCL scaffolds with spatiotemporal delivery of CTGF andTGFβ3 successfully formed native-like micro-scale multi-tissueinterfaces, evidencing the potential to regenerate multi-tissue complexsuch as tendon-bone insertion, synovial joints, fibrocartilaginoustissues, and periodontium.

Example 3: Spatiotemporal Delivery of Growth Factors Embedded in 3DPrinted Multi-Tissue Scaffolds

The following example will test the efficacy of the spatiotemporaldelivery of growth factors (GFs) embedded in 3D printed scaffolds (seee.g., Example 1) for regeneration of multi-tissue complexes.

Scaffolds are first cultured with multiple stem/progenitor cellpopulations in vitro (see e.g., Example 5, Example 6, and Example 7),followed by in vivo implantation in a relevant animal model (see e.g.,Example 5).

Scaffolds are designed with spatiotemporal delivery of growth factors.An anterior cruciate ligament-fibrocartilage-bone interfaces (see e.g.,FIG. 4A) and supraspinatus tendon-fibrocartilage (unmineralized andmineralized)-bone interfaces (see e.g., FIG. 4B) are reconstructed in3D-printed scaffolds with spatiotemporally delivered growth factors.

In a similar way, a cementum (CM)-periodontal ligament (PDL)-alveolarbone (AB) complex is reconstructed (e.g., periodontium) (see e.g., FIG.4C). The width of each interface component, matrix type, internalmicrostructure (e.g. pattern, fiber size, or channel width), or deliveryof relevant growth factors is applied in an integrated scaffold.

In addition to multi-tissue interfaces, the described spatiotemporaldelivery system embedded in 3D-printed scaffolds is applied forrecapitulating the gradient matrix distribution and organization ininhomogeneous multiphase tissues, such as TMJ disc and knee meniscus(see e.g., FIG. 4D).

Histology presented in FIG. 4 was adopted from Lu and Thomopoulos 2013Annu Rev Biomed Eng. 15, 201-26.

Example 4: Spatiotemporal Delivery of Growth Factors Embedded in 3DPrinted Rotator Cuff Scaffolds

The following example will test the efficacy of the spatiotemporaldelivery of growth factors (GFs) embedded in 3D printed scaffolds (seee.g., Example 1) for regeneration of multi-tissue complexes.

Scaffolds are designed with spatiotemporal delivery of growth factors. Asupraspinatus tendon (i.e., rotator cuff) to-bone interface (see e.g.,FIG. 6 ) measuring 40 mm×40-70 mm×0.5-1 mm is reconstructed in3D-printed scaffolds with spatiotemporally delivered growth factorsCTGF, CTGF+TGFβ3, and BMP2 and implanted at the tendon to bone interfacefor rotator cuff regeneration. Example 7 demonstrates in vitroregeneration of rotator cuff (supraspinatus tendon)-to-bone interfacesbetween tendon, fibrocartilage, and bone (see e.g., FIG. 20 ). FIG. 21also shows immunofluorescence images of tendon-to-bone scaffoldregenerating tendon-to-bone complex.

Example 5: In Vitro and In Vivo Formation of TMJ Scaffolds Mimic NativeMultiphase Fibrocartilage

The following example provides for a 3D printed biocompatible scaffoldembedded with a spatiotemporal delivery of CTGF and TGFβ3 encapsulatedin microspheres.

A micro-precise spatiotemporal delivery system embedded in 3D printedscaffolds was developed. PLGA microspheres (μS)-encapsulated with growthfactors (GFs) were embedded inside PCL microfibers that constitutecustom-designed 3D scaffolds. Given the substantial difference in themelting points between PLGA and PCL and their low heat conductivity, themicrospheres were able to maintain its original structure whileprotecting the GF's bioactivities. Micro-precise spatial control ofmultiple GFs was achieved by interchanging dispensing cartridges duringa single printing process. Spatially delivered GFs with a prolongedrelease guided formation of multiple tissues or micro-width multi-tissueinterfaces from bone marrow derived mesenchymal stem/progenitor cells(MSCs).

To investigate efficacy of the micro-precise delivery system embedded in3D printed scaffold, temporomandibular joint (TMJ) disc scaffolds werefabricated with micro-precise spatiotemporal delivery of CTGF and TGFβ3,mimicking native-like multiphase fibrocartilage. In vitro, TMJ discscaffolds spatially embedded with CTGF/TGFβ3-μS resulted in formation ofmultiphase fibrocartilaginous tissues from MSCs. In vivo, TMJ discperforation was performed in rabbits, followed by implantation ofCTGF/TGFβ3-μS embedded scaffolds. After 4 weeks, CTGF/TGFβ3-μS embeddedscaffolds significantly improved healing of the perforated TMJ disc ascompared to the degenerated TMJ disc in the control group with scaffoldembedded with empty μS. In addition, CTGF/TGFβ3-μS-embedded scaffoldssignificantly prevented arthritic changes on TMJ condyles. It was foundthat micro-precise spatiotemporal delivery system embedded in 3Dprinting can serve as an efficient tool to regenerate complex andinhomogeneous tissues.

TMJ Disc.

TMJ disc is featured by its region-dependent distribution and/ororientation of collagen fibers and cartilaginous matrix includingcollagen type II, proteoglycan, and glycosaminoglycan (GAGs). Collagenis the predominant extracellular matrix (ECM) in a TMJ disc, with itsdense fibrous structure aligned circumferentially in the peripheral ringand predominantly aligned in anteroposterior direction in theintermediate zone (Allen and Athanasiou 2006a; Kalpakci et al. 2011;Scapino et al. 1996; Willard et al. 2012). Collagen alignment indifferent regions is primarily attributed to the region-dependentanisotropic tensile properties of TMJ disc (Allen and Athanasiou 2006a;Kalpakci et al. 2011; Scapino et al. 1996). GAGs, a key component ofcartilage, are primarily localized in the intermediate zone and itsinterfaces with anterior/posterior band. Despite the low content (˜5%),GAGs can be associated with the regional variance in viscoelasticproperties under compression (Lumpkins and McFetridge 2009; Willard etal. 2012). Cell populations in TMJ disc can also be heterogeneous, as70% are fibroblast-like cells throughout the tissues and 30% arechondrocyte-like cells localized in the intermediate zone (Allen andAthanasiou 2006a; Detamore et al. 2006). The heterogeneous biochemicalcomposition/orientation and the associated anisotropic mechanicalproperties can be a challenge to TMJ disc regeneration.

TMJ disorders (TMJDs) affect over 10 million Americans and an annualcost for the treatment is estimated at ˜$4B, per NIDCR. TMJ disc is abiconcave fibrocartilaginous tissue positioned between mandibularcondyle and glenoid fossa, and its displacement or ‘internalderangement’ is highly correlated with onset and progress of TMJDs.Discectomy has been often performed in patients with damaged and/orseverely displaced TMJ disc to alleviate symptoms, with yetcontroversial experimental and clinical outcome. Synthetic oralloplastic disc replacements have also failed to reduce pain andrestore joint function, frequently leading to further jointdegeneration. Accordingly, the scaffolds and methods herein (e.g., TMJdisc regeneration) overcome limitations of the current treatments forligament disorders, such as TMJ disorders.

Materials and Methods.

(i) Preparation of PLGA μS Encapsulated with GFs

Poly(d-l-lactic-co-glycolic acid) (PLGA) with a PLA/PGA ratio of 75:25was purchased from Sigma (St. Louis, Mo.). PLGA microspheres (μS)encapsulating recombinant human bone morphogenetic protein 2 (BMP2),connective tissue growth factor (CTGF) and transforming growth factor(TGFβ3) were prepared by a modified double-emulsion technique, awell-established control-delivery vehicle demonstrating preservedbioactivity of GFs. Briefly, 500 mg PLGA was dissolved into 5 mLchloroform followed by adding 250 μL diluted growth factor (GF). Thissolution was then 146 emulsified (primary emulsion) by ultrasonicatingfor 5 minutes. The primary emulsion (w/o) was then added to 10 mL 4%(w/v) PVA (poly vinyl alcohol) solution to form the second emulsion(w/o/w) by 2 minutes ultrasonication followed by 1 minute vortexing.This double emulsion solution was then added to 250 mL of 0.3% PVAsolution followed by continuous stirring for 2 hours to evaporate thesolvent. Finally, the microspheres (μS) were filtered, washed with DIwater, resuspended in DI water and then lyophilized. The size of PLGA μSwere analyzed using SEM (ZEISS SUPRA 55VP). To confirm the embedding anddistribution of μS, dextran conjugated with rhodamine A and dextranAlexa Fluor® 488 (Invitrogen, Eugene, Oreg., USA) were encapsulated inPLGA μS. For GFs encapsulated PLGA μS preparation, 500 mg PLGA was usedfor each of 5 μg BMP2, 2.5 μg TGFβ3, and 10 μg CTGF, respectively. DIwater in the same volume was used for preparing empty μS.

(ii) Fabrication of 3D Printed Scaffolds with Spatiotemporal Delivery ofMultiple GFs

Scaffolds embedded with micro-precise spatiotemporal delivery systemwere made by layer-by-layer deposition technique using 3D Bioplotter®(4th generation; EnvisionTec, Germany). Polycaprolactone (PCL)(Polyscience Inc., Warrington, Pa., USA) powder and PLGA-μS were mixed(50 mg μS per 1 g PCL) together and filled into a high temperaturecartridge. The cartridge was then heated up to 120° C. and μS-embeddedPCL microfibers were dispensed through a micro-needle. Given the highmelting point of PLGA over 200° C. and the low heat conductivity, theoriginal structure of PLGA μS and the bioactivity of encapsulated GFswere maintained during the printing process, as described in the resultsection below. Microfibers strand diameter was controlled by the needleinternal diameter (ID). Fluorescence images were taken using an OlympusIX73 microscope (Center Valley Pa., USA) and Maestro™ in-vivofluorescence imaging system (Cambridge Research & Instrumentation, Inc.(CRi); Woburn, Mass., USA).

Cylindrical scaffolds (5 mm diameter and 5 mm height) with and withoutμS and single layered square shape (5×5 mm) scaffolds with GFsencapsulated μS were 3D printed for mechanical test and releasekinetics, respectively. Compressive modulus and ultimate strength withor without μS were measured at a constant compression rate (1%/min)using an ElectroForce BioDynamic Testing system (Bose Corp., EdenPrairie, Minn., USA). To check if a surface treatment, creatingmicro/macro pores and cracks, can increase the release rate of the GFdelivered in 3D printed scaffolds via μS embedding, these scaffolds weresubjected to NaOH (6M) treatment for 4 hours. Morphological and/orstructural changes in scaffolds after NaOH treatment were observed underSEM. The difference in release rate between untreated and NaOH treatedscaffolds measured by incubating BMP2 encapsulated μS-embedded PCLscaffolds for 56 days in PBS buffer at 37° C.

(iii) MSCs Differentiation and Multi-Tissue Formation in GF/μS-EmbeddedPCL Scaffolds

For multi-tissue formation through MSCs differentiation, scaffolds witha dimension of 5 mm×5 mm×0.5 mm were fabricated with 400 μm microstrandsand 600 μm inter-strand distance. CTGF, TGFβ3 and BMP2 μS (50 mg/1 gPCL) was delivered in scaffolds to stimulate fibroblastic, chondrogenic,and osteogenic differentiation, respectively. Empty μS embedded group(−GF) served as control. MSCs were isolated from human bone marrow(AllCells, Alameda, Calif.) following an established protocol. P3-5 MSCsat a density of 1×10⁶ cells/mL were seeded into the scaffolds byinfusing the cell suspended fibrin gel (20 mg/ml fibrinogen+20 U/mlthrombin) into microchannels of the scaffolds followed by 1 hourincubation at 37° C. for gel formation. Culture medium (DMEMsupplemented with 10% FBS and 1% antibiotics) was added immediatelyafter gel formation, and cultured for 4 weeks with media changes everyother day. Differentiation medium was prepared. Briefly, low glucoseDMEM was supplemented with 100 nM dexamethasone, 10 mMβ-glycerophosphate and 0.05 mM ascorbic acid-2-phosphate for osteogenicdifferentiation. Ascorbic acid (50 μg/mL) was added to the low glucoseDMEM to support fibrogenic differentiation. High glucose DMEM with 1%FBS was supplemented with 1% ITS+1 (Sigma) for chondrogenicdifferentiation. After 4 weeks culture, the cell seeded scaffolds werefixed in formalin, sectioned in 5 μm thickness, and stained withPicrosirius red (PR), Safranin O (SO), and Alizarin red (AR).

(iv) Engineering Rabbit TMJ Disc with Anatomically GF/μS-Embedded TMJDisc Scaffolds

To test the hypothesis that multi-tissue interface can be formed in asingle scaffold by spatially delivered multiple growth factors,single-layered scaffolds (1.5 mm×1.5 mm) were fabricated with paralleloriented PCL microstrands (100 μm) and inter-strand spaces (100 μm),alternatingly embedding CTGF and TGFβ3 μS by changing the dispensingcartridge during the printing process. The combination of CTGF and TGFβ3were selected given their unique role to induce fibrocartilaginousdifferentiation of MSCs. Then human bone marrow MSCs (1×10⁶ cells/mL)were seeded as described previously, followed by culturing with 1:1mixture of fibrogenic and chondrogenic supplements. After 6 weeks,harvested constructs were fixed and sectioned for histological analysiswith PR, AB and immunofluorescence for collagen type I and II.Fluorescence images were taken using Maestro™ imaging system and OlympusIX73 microscope. To engineer rabbit TMJ disc, a scaffold was designedwith the anatomic shape and dimension of the native TMJ disc from a 3Dlaser-scanned contour. Region specific internal architecture of thescaffold was designed to recreate the region-dependent collagenorientation. Briefly, PCL microstrands (200 μm) were orientedcircumferentially in the anterior and posterior bands, while oriented inthe anteroposterior direction in the intermediate zone with 100-200 μmof interstrand spaces. Both CTGF and TGFβ3 encapsulated μS were embeddedin the microfiber strands in intermediate zone, whereas anterior andposterior zones were only embedded with CTGF encapsulated μS. Then MSCs(2×10⁶ cells/mL) were seeded as described previously and cultured for 6weeks with 1:1 mixture of fibrogenic and chondrogenic supplements.Fibrocartilaginous tissue formation was evaluated by histologicalanalysis as described above.

(v) In Vivo Implantation of GF/μS-Embedded TMJ Disc Scaffolds

All the animal procedures were followed by IACUC approved protocol withskeletally mature New Zealand White rabbits (3.5-4.0 kg in body weight).Briefly, rabbits were sedated with ketamine (35 mg/ml)/xylazine (5mg/ml) cocktail, and anesthesia was maintained by 1-5% isofluoraneinhalation. Rabbits were placed in the right lateral position to exposethe left TMJ on a warm-water flowing heating pad to maintain bodytemperature. The TMJ region, just posterior to the orbital cavities, wasshaved and prepped with povidone iodine/ethanol and draped in a sterilesurgical manner. The surgical site was then injected with a localanesthetic, 2% lidocaine with 1:200,000 epinephrine, to minimizediscomfort. A 2-3 cm vertical incision between the posterior of thelateral orbital wall and the external acoustic meatus were performed toexpose the TMJ disc followed by making a 2.5 mm punch on the disc usinga sterile puncher. Pre-made sterile 3D-Printed (3DP) scaffolds in a discshape with a dimension of 2.5×0.5 mm (diameter×thickness) were implantedusing vicryl suture. These scaffolds had CTGF and TGFβ3 μS mixtureconsisted of 200 μm microstrands and 200-400 μm interstrand spaces.Scaffolds with empty μS served as control. Upon implantation ofscaffolds, the skin was sutured continuously with non-absorbable nylonsutures. At 4 weeks post-surgery, rabbits were euthanized by IVinjection of a lethal dose of euthasol (100 mg/kg). TMJ discs, condylesand glenoid fossa were harvested after 4 weeks, followed byhistomorphological analyses.

(vi) Statistics

For all the quantitative data, following confirmation of normal datadistribution, one-way analysis of variance (ANOVA) with post-hoc TukeyHSD tests was used with p value of 0.05. Sample sizes for allquantitative data were determined by power analysis with one-way ANOVAusing an a level of 0.05, power of 0.8, and effect size of 1.50 chosento assess matrix synthesis, gene expressions, and mechanical propertiesin the regenerated meniscus tissues and controls upon verification ofnormal data distribution.

Results.

(i) Micro-Precise Spatiotemporal Delivery of Multiple Growth Factors in3D Printed PCL Scaffolds

PCL slurry mixed with PLGA μS-encapsulated with dextran conjugated withAlexa Fluor® 488 or Rhodamine was successfully dispensed through astainless steel microneedle (100-400 μm of inner diameter) at 120° C.(see e.g., FIG. 1A). Fluorescence microscopic images showed that PLGA μSwere embedded inside PCL microstrands, as maintaining their originalspherical structure (see e.g., FIG. 1B). Successful embedding of thePLGA μS in the 3D-deposited PCL microstrands were confirmed usingfluorescent dextran/μS. Given the PLGA's melting point over 200° C., μSare able to maintain their original structure during the dispensingprocess, as protecting encapsulated GFs (see e.g., FIG. 1B). Thisapproach enables the fabrication of 3D scaffolds with multiple growthfactors delivered at desired location by changing dispending cartilageduring the printing process, leading to fully integrated 3Dcustom-designed scaffold (see e.g., FIG. 1B). Using fluorescentdextran/μS, it was confirmed that PLGA μS were successfully embedded inthe 3D-deposited PCL microstrands, with custom designed scaffoldstructure/pattern (see e.g., FIG. 1C). To facilitate seamless dispensingof PCL blend through micro-needles (50-400 μm in I.D.), the diameter ofPLGA μS were reduced by 23±14 μm (see e.g., FIG. 7A-FIG. 7C).

By applying ultrasonication in the double emulsion technique, PLGA μSwere prepared in diameters of 22.68±14.89 μm that is sufficiently smallto facilitate 3D printing process (see e.g., FIG. 7A-FIG. 7C). There wasno significant differences in compressive modulus and ultimate strengthbetween PCL alone and PLGA μS-embedded PCL scaffold (50 mg per 1 g ofPCL) (see e.g., FIG. 7D) (n=5 per group). NaOH (6 M) treatment for 4hours induced micro-pores and cracks on the surface of PCL microstrands(see e.g., FIG. 7E), which led to accelerated release of encapsulatedBMP-2 as compared to untreated scaffold (see e.g., FIG. 7F).

Fluorescence images of whole scaffolds from Maestro™ imaging system(PerkinElmer Inc., Waltham, Mass.) showed evenly distributed μS in 3Dprinted structure (see e.g., FIG. 1D, FIG. 1D).

Mechanical properties of 3D printed scaffolds, including compressivemodulus and ultimate strength, were not significantly altered byembedding 50 mg PLGA μS per 1 g in PCL (see e.g., FIG. 7D). Byinterchanging dispensing cartridges during a printing process, μSencapsulating different dextrans were successfully embedded in desiredlocations in an integrated 3D PCL structure (see e.g., FIG. 1D). CTGF,TGFβ3, and BMP-2 delivered in PCL scaffolds via μS embedding showed asustained release up to 42 days in vitro (see e.g., FIG. 1E). Detectionof released GFs by ELISA further confirm the preserved biochemicalstructure of the delivered GFs. Surface treatment with 6 M NaOH for 4hours resulted in micro-pores on the surface of PCL microstrands (seee.g., FIG. 7E) which in turn significantly accelerated the release ratea growth factor embedded in PCL scaffold via μS embedding in comparisonwith no surface treatment (see e.g., FIG. 7F).

(ii) MSC Differentiation Guided in GF/μS-Embedded Scaffolds

After 4 weeks culture with MSCs (1 M cells/ml via fibrin gel),GF/μS-embedded scaffolds guided formation of multiple types of tissuesdepending on the delivered growth factor (see e.g., FIG. 8A-FIG. 8L).

Bioactivity and multi-tissue formation was observed at 4 weeks culturewith hMSCs (P4—5, 2 M/mL via collagen gel).

After 4 weeks culture with MSCs, Picrosirius Red (PR) staining (FIG.8A-FIG. 8D) show dense collagenous matrix formed in CTGF μS-embeddedscaffolds (see e.g., FIG. 8B) as compared to BMP-2 (see e.g., FIG. 8D),TGFβ3 (see e.g., FIG. 8C), and control groups (see e.g., FIG. 8A, FIG.8C, and FIG. 8D).

Similarly, Safranin O (Saf O) staining (see e.g., FIG. 8E-FIG. 8H)showed cartilaginous tissue was formed in TGFβ3 μS-embedded scaffolds(see e.g., FIG. 8G), whereas Alizarin red (AR) staining (see e.g., FIG.8I-FIG. 8L) showed mineralized matrix formed in BMP-2 μS-embeddedscaffolds. For example, TGFβ3-delivered scaffolds led to formation ofSaf O-positive cartilaginous matrix (see e.g., FIG. 8H), whereas BMP-2delivery resulted in Alizarin red (AR)-positive mineralized tissues (seee.g., FIG. 8J), as compared to the control and the other growth factorgroups (see e.g., FIG. 8E-FIG. 8G, FIG. 8I, FIG. 8K-FIG. 8L).

The induced differentiation of MSCs in the GF/μS-embedded scaffolds (seee.g., FIG. 8 ) confirms the preserved bioactivities of the deliveredGFs.

(iii) Formation of Multi-Tissue Interfaces by Alternatingly DeliveredCTGF and TGFβ3

Single-layered rectangular scaffolds (1.5 mm×1.5 mm) were fabricatedwith parallel oriented PCL microfibers (100 μm) and inter-fiberschannels (100 μm) (see e.g., FIG. 9A). The PCL microfibers werealternatingly embedded with CTGF and TGFβ3 μS (see e.g., FIG. 9B). Thesize of microfibers and inter-fibers space was 100 μm. Human MSCs weredelivered in the scaffold's inter-fiber space via fibrin gel.

After 4 weeks culture with MSCs/fibrin delivered in the inter-strandsmicrochannels, immunofluorescence and histology revealed integratedinterfaces of COL-I+ and COL-II+ matrices within the −100 μmmicrochannels (see e.g., FIG. 9B, FIG. 9C, FIG. 9D, and FIG. 9E),corresponding to the alternatingly located CTGF and TGFβ3 (see e.g.,FIG. 9B).

(iv) Rabbit TMJ Disc Engineering with 3D Printed Scaffolds EmbeddingCTGF and TGFβ3 μS

PR and AB staining demonstrated the anisotropic collagen orientation andregionally variant fibrocartilaginous matrix in native rabbit TMJ discs(see e.g., FIG. 10A). Collagen was distributed throughout the TMJ discwith circumferential and anteroposterior alignment in peripheral bandsand intermediate zone, respectively (see e.g., FIG. 10A). AB-positivefibrocartilaginous matrix and rounded chondrocyte-like cells werepredominantly distributed in the intermediate zone and its interfacewith the peripheral bands (see e.g., FIG. 10A). From a 3D laser-scannednative contour data, anatomically correct rabbit TMJ disc scaffolds werefabricated with 100 μm PCL strands and inter-strands microchannels (seee.g., FIG. 10B). PCL microstrands were oriented following theregion-dependent collagen alignment. CTGF μS were embedded throughoutthe scaffolds and TGFβ3 μS were embedded in the intermediate zone.Rhodamine μS was used to show a representative distribution of μS in theTMJ disc scaffolds (see e.g., FIG. 10B). After 6 weeks culture with MSCseither from human bone marrow or rabbit TMJ synovium, multiphasefibrocartilaginous tissue with densely aligned fibrous matrix wasobserved in the anterior and posterior bands and fibrocartilaginousmatrix in the intermediate zone (see e.g., FIG. 10C).

(v) Improved Healing of TMJ Disc by 3D Printed Scaffolds withSpatiotemporal Delivery of CTGF and TGFβ3

At 6 weeks in vivo, implantation of 3D printed scaffolds embedded withCTGF and TGFβ3 μS successfully improved healing of perforated rabbit TMJdisc. GF/μS-embedded scaffolds resulted in full recovery of theperforated defects with spatially distributed fibrocartilaginous matrix,reminiscent of native TMJ disc (see e.g., FIG. 11A). However, scaffoldswithout GF led to severe disc degeneration with structural breakdown(see e.g., FIG. 11A). High magnification histology demonstrated that therounded chondrocyte-like cell population on the surface of native TMJdisc was successfully reconstructed in the regenerated TMJ disc withGF/μS-embedded scaffolds (see e.g., FIG. 11B). Degenerating TMJ discwithout GF showed the loss of chondrocyte-like cells, replaced byspindle shaped cells within a relatively loose fibrous matrix (see e.g.,FIG. 11B).

Consistently, there was no sign of cartilage defects on the articularsurface of TMJ condyle, while scaffold without growth factor resulted insome vertical erosions in cartilage, as compared to native articularcartilage (see e.g., FIG. 12A-FIG. 12C). Quantitatively, the cartilagethickness with no growth factor was significantly thinner than nativeand the GF/μS-embedded scaffolds (see e.g., FIG. 12D). Similarly, OARSIosteoarthritis score was significantly lower with GF/μS-embeddedscaffolds than scaffolds without GF (see e.g., FIG. 12E). The glenoidfossa showed a dense Saf O-positive cartilage layer when GF/μS-embeddedscaffolds as compared to the native with no noticeable sign of cartilagedefects (see e.g., FIG. 13A-FIG. 13C).

Discussion.

This study demonstrates an important advancement in 3D printing oftissue engineering scaffolds as a control-delivery vehicle for bioactivecues. This study showed the achievement of micro-precise delivery ofmultiple growth factors in different locations in a 3D printed scaffoldwith a sustained release over time. The novel 3D printed scaffoldembedded with PLGA μS encapsulating CTGF, TGFβ3 and/or BMP-2successfully led to formation of multiphase tissue constructs byinducing region-specific differentiation of MSCs in a native-levelresolution. In addition, profibrogenic CTGF and chondrogenic TGFβ3 weresuccessfully delivered in 3D printed anatomically correct TMJ discscaffolds in a spatiotemporal manner, mimicking multiphase distributionof fibrocartilaginous matrix in native tissue. Upon in vivoimplantation, TMJ disc scaffolds embedded with CTGF/TGFβ3-μSsignificantly improved healing of perforated TMJ disc in rabbits withnative-like multiphase fibrocartilaginous tissue and prevented arthriticchanges in the articular surfaces.

A control group with defect only, a likely representative of TMJperforation under severe TMJ disorders is not shown. Although it is notshown in this study, the identical surgical model of 2.5 mm TMJ discperforation without a treatment, performed by the same surgeon, showed afailure of disc healing and severe arthritis that validates the in vivostudy design to investigate effects of spatiotemporally delivered GFs.Given the surgical difficulty to reconnect whole disc in rabbits, apartial graft was implanted after disc perforation. In addition, theanimal model may represent or model TMJ disc perforation caused byprolonged and severe disc dislocation but osteoarthritis.

PCL degradation by hydrolysis is relatively slow. Interestingly, the invivo implanted PCL based 3D printed scaffolds with and without GFdelivery were fully degradated and replaced by newly formed tissues by 4weeks. There are several potential explanations for the accelerated invivo degradation, including high surface-to-volume ratio of scaffoldsconsisted repeats of microstrands and interstrands and the alteredintrasynovial biochemical environment upon TMJ disc injury. More likely,embedding PLGA μS within PCL microfibers may increase the degradationrate. The in vitro degradation study in NaOH, PCL scaffold embedded withPLGA μS (50 mg/l g PCL) exhibited significantly accelerated degradationin comparison with PCL alone (data not shown).

In conclusion, the novel approach to spatiotemporally deliver multiplegrowth factors in 3D printed scaffolds may represent an efficient toolto develop ready-to-implant bioscaffolds guiding regeneration of complexinhomogeneous tissues. The micrometer-scale resolution in spatialcontrol of sustainably releasing GFs and the customized microstructurein 3D printed scaffolds will also be applicable for stem cell-basedregeneration of inhomogeneous tissues and multi-tissue complex,including knee meniscus, tendon/ligament-to-bone interfaces, andperiodontium.

Example 6: In Vitro Formation of TMJ Scaffolds

The following example provides for a 3D printed biocompatible TMJscaffold embedded with a spatiotemporal delivery system (i.e.,microspheres) encapsulating CTGF and TGFβ3 mimicking native multi-tissuecomplex.

This example shows the development of 3D-printed anatomically correctscaffolds with region-variant microstrands alignment, mimickinganisotropic collagen alignment in TMJ disc and corresponding mechanicalproperties. Connective tissue growth factor (CTGF) and transforminggrowth factor beta 3 (TGFβ3) were delivered in the scaffolds byspatially embedding CTGF or TGFβ3-encapsulated microspheres (μS) toreconstruct the regionally variant fibrocartilaginous matrix in nativeTMJ disc. When cultured with human mesenchymal stem/progenitor cells(MSCs) for 6 weeks, 3D-printed scaffolds with CTGF/TGFβ3-μS resulted ina heterogeneous fibrocartilaginous matrix with overall distribution ofcollagen-rich fibrous structure in the anterior/posterior (AP) bands andfibrocartilaginous matrix in the intermediate zone, reminiscent ofnative TMJ disc. High dose of CTGF/TGFβ3-μS (100 mg μS/g of scaffold)showed significantly more collagen II and aggrecan in the intermediatezone than a low dose (50 mg μS/g of scaffold). Similarly, high dose ofCTGF/TGFβ3-μS yielded significantly higher collagen I expression in theAP bands as compared to the low dose and empty μS. From stressrelaxation tests, the ratio of relaxation modulus to instantaneousmodulus was significantly smaller with CTGF/TGFβ3-μS than empty μS.Similarly, a significantly higher coefficient of viscosity was achievedwith the high dose of CTGF/TGFβ3-μS as compared to the low dose andempty μS, suggesting the dose-effect of CTGF and TGFβ3 on fibrocartilageformation. This example shows an efficient approach to engineering TMJdisc graft with anisotropic scaffold microstructure, heterogeneousfibrocartilaginous matrix, and region-dependent viscoelastic properties.

In this study, a 3D printing technique and spatiotemporal deliverysystem was developed to construct an anatomically correct TMJ discscaffold with regionally variant microstructure and the associatedmechanical properties. The scaffolds with spatiotemporal delivery ofCTGF and TGFβ3 successfully induced the region-dependentfibrocartilaginous differentiation of MSCs, consequently leading toformation of heterogeneous fibrocartilage reminiscent of native TMJdisc. Dose effects of CTGF and TGFβ3 on the viscoelastic properties ofthe engineered TMJ disc were also investigated with an ultimate goal ofdeveloping a ready-to-implant TMJ disc biograft.

Materials and Methods.

(i) Fabrication of 3D-Printed TMJ Disc Scaffolds with SpatiotemporalDelivery of CTGF and TGFβ3

Anatomically correct TMJ disc scaffolds were fabricated withpolycaprolactone (PCL) using layer-by-layer deposition technique with 3DBioplotter® (4th generation; EnvisionTec, Germany) according to Lee etal. 2014. Briefly, the anatomical shape and dimension of TMJ disc wereadopted from 3D laser-scanned contour that was reconstructed into a 3DCAD model (see e.g., FIG. 14A), followed by 3D printing scaffolds. TMJdisc scaffolds were constructed with repeating microstrands andinterstrand microchannels with their orientation predominantly in thecircumferential and anteroposterior directions in the peripheral ringand intermediate zone, respectively (see e.g., FIG. 14B), mimickingnative anisotropic collagen alignment. The size of PCL microstrands (300μm) and microchannels (300 μm) and the relative density of microstrandsparallel to versus perpendicular to the alignment direction (2:1) weredetermined to closely approximate the tensile properties of those ofnative disc in the circumferential and anteroposterior directions,respectively (see e.g., FIG. 14C).

(ii) Spatiotemporal Delivery of CTGF and TGFβ3

Because a combination of profibrogenic CTGF and chondrogenic TGFβ3growth factors induce fibrochondrogenic differentiation of MSCs, CTGFand TGFβ3 were spatiotemporally delivered into the 3D-printed scaffoldsto guide formation of heterogeneous fibrocartilage reminiscent of nativeTMJ disc. For a prolonged release, CTGF and TGFβ3 were firstencapsulated in 75:25 poly(lactic-co-glycolic acids) (PLGA) microspheres(μS) by double-emulsion technique, according to Lee et al. 2010b and Leeet al. 2014. A single dose of CTGF (10 μg) and TGFβ3 (5 μg) wasencapsulated in 500 mg PLGA μS according to Lee et al. 2010b and Lee etal. 2014. CTGF or TGFβ3 encapsulated PLGA μS were then mixed with PCLpowder (Polyscience Inc., Warrington, Pa.) (50 mg or 100 mg μS per 1 gPCL) in a high temperature dispensing cartridge of 3D-Bioplotter®. Thedoses of CTGF and TGFβ3 encapsulation and PLGA μS were pre-optimized.The cartridge was then heated up to 120° C. and μS-embedded PCLmicrofibers were dispensed through a micro-needle. Given the PLGA's highmelting point over 200° C. and low heat conductivity (Qian et al. 2001),PLGA μS were embedded in PCL microstrands constructing 3D scaffoldswhile maintaining their original structure and preserving encapsulatedgrowth factors (GF) (Tarafder et al. 2016). CTGF μS-embedded and CTGF &TGFβ3 μS-embedded PCL microstrands were constructed into the peripheralbands and the intermediate zone, respectively, to recapitulate thefibrous matrix in the peripheral bands and fibrocartilaginous matrix inthe intermediate zone. To confirm the spatial delivery of GFs, dextranconjugated with Alexa Fluor® 488 and 546 were encapsulated in PLGA μS,followed by imaging with Maestro™ in-vivo imaging system (CRi; Woburn,Mass., USA). In vitro release kinetics of the delivered GFs weremeasured by ELISA up to 42 days by incubating the scaffolds in PBS or0.1% BSA buffer at 37° C. with gentle agitation, as per Lee et al. 2010band Lee et al. 2014.

(iii) Human TMJ Disc Engineering with MSCs

Human mesenchymal stem/progenitor cells (MSCs) were isolated fromcommercially available, fresh whole bone marrow samples of anonymousadult donors (AllCells, Alameda, Calif.) (age range: 20-25 years old)according to Lee et al. 2009; Lee et al. 2010b; and Lee et al. 2014.Passage 3/4 MSCs (2 M cells/mL) were seeded in the 3D-printed TMJ discscaffolds by infusing the cell suspended neutralized collagen I solution(2 mg/mL) into the scaffold's microchannels, followed by 30 minutesincubation at 37° C. for gel formation. MSC-seeded scaffolds were thencultured for 6 weeks with 1:1 mixture of fibrochondrogenic inductionsupplement (FIS) (50 μg/mL ascorbic acids) and chondrogenic inductionsupplements (CIS) (1% 1×ITS+1 solution, 100/ml sodium pyruvate, 50 μg/mlL-ascorbic Acid 2-phosphate, 40 μg/ml L-proline, 0.1 μM dexamethasone)(Lee et al. 2014). Low and high doses (50 mg and 100 mg μS per 1 g PCL)of CTGF/TGFβ3 μS in the 3D-printed scaffolds were tested for TMJ discengineering with MSCs. Low and high doses of empty μS were applied ascontrols. Harvested samples after 6 weeks were analyzed using histologywith Safranin O (Saf-O) and Picrosirius red (PR) (Lee et al. 2010b; Leeet al. 2014). Total collagen and GAGs contents were measured usingBiocolor assay kits (Carrickfergus, UK) following Lee et al. 2010b.Immunofluorescence was performed to evaluate collagen I and II (COL-I &II) and aggrecan (AGC) as per Lee et al. 2014. Relative areas of COL-IIand AGC positive matrix in the anterior and posterior (AP) bands andintermediate zone were calculated using an imaging process following Leeet al. 2010a. Expression of COL-I mRNA was measured by qRT-PCR (Lee etal. 2010a; Lee et al. 2014) for the MSC-seeded scaffolds with low andhigh doses of CTGF/TGFβ3 μS and empty μS.

(iv) Mechanical Property Tests

Mechanical properties of 3D-printed scaffolds and the engineered TMJdisc were evaluated by compression, tensile, and stress relaxation testsas per Lee et al. 2010a and Lee et al. 2014 using BioDynamics® testingsystem (TA instruments, New Castle, Del.). For tensile tests, scaffoldswere prepared in a dog bone shape with length of 25 mm and averagethickness of 1 mm. After preconditioning of 15 cycles of 0-10% strain,tensile tests were performed at 1% strain/min. The linear portion ofeach stress-strain curve was used to determine the tensile modulus. Forcompression and stress relaxation tests, disc-shaped samples (5×2 mm²)were prepared from the AP bands and intermediate zone of the TMJ discconstructs. Using unconfined compression, samples were preconditionedwith 15 cycles of 0-10% compressive strain, followed by compression at1% strain/min. For stress relaxation, 30% step strain was applied andheld until a relaxation plateau was reached. A Kelvin standard solidviscoelastic model was applied to calculate instantaneous modulus (Ei),modulus of relaxation (Er), and coefficient of viscosity (μ) usingMATLAB curve fitting tool as per Lee et al. 2014. The mechanicalproperties of engineered TMJ discs were compared with those of nativeTMJ discs (18˜65 year-old) from National Disease Research Interchange(NDRI).

(v) Statistical Analysis

Upon confirmation of normal data distribution, One-way ANOVA with apost-hoc Tukey test were used to compare between the groups with p value<0.05 considered significant.

Results.

(i) Optimal Microstructure of Scaffolds to Achieve Initial TensileProperties

Tensile properties of scaffolds were affected by sizes of microstrandsand microchannels. Among the multiple configurations of microstructure,300 μm PCL microstrand diameter and 300 μm microchannels with 2:1 ratioof parallel microstrands and perpendicular microstrands showed thetensile modulus of scaffolds most approximately to that of native TMJdiscs (see e.g., FIG. 14C).

(ii) Spatiotemporal Delivery of CTGF and TGFβ3 in 3D-Printed TMJ DiscScaffolds for Generation of Heterogeneous Fibrocartilage

Representative fluorescence images with μS-encapsulating Alex Fluora®488 and 546 demonstrated that CTGF is delivered throughout the scaffoldswhereas CTGF and TGFβ3 are delivered in the intermediate zone of the TMJdisc scaffolds (see e.g., FIG. 14D). CTGF and TGFβ3 delivered in thescaffold showed a sustained release up to 42 days in vitro (see e.g.,FIG. 14E). When cultured with MSCs (2 M/mL) for 6 weeks, scaffolds withspatiotemporal delivery of CTGF and TGFβ3 formed heterogeneousfibrocartilaginous tissues, featured by COL-I+ fibrous matrix throughoutthe scaffolds and COL-I+/AGC+ fibrocartilaginous matrix localized in theintermediate zone (see e.g., FIG. 14G) in comparison with empty μS (seee.g., FIG. 14F). Quantitative matrix assays demonstrated that totalcollagen contents per wet weight were significantly higher both in theintermediate zone (IZ) and the anterior/posterior band (AP) with GFdelivery as compared to control without GF (see e.g., FIG. 14H) (p<0.05;n=5 per group). Similarly, GAGs content was significantly higher with GFdelivery as compared to control (see e.g., FIG. 14I) (p<0.05; n=5 pergroup). IZ showed significantly higher GAGs content than AP (see e.g.,FIG. 14I) (p<0.05; n=5 per group).

(iii) Dose Effect of CTGF and TGFβ3-μS in TMJ Disc Tissue Engineering

After 6 weeks culture with MSCs, growth factors (GF; CTGF and TGFβ3)encapsulated μS-embedded scaffolds formed heterogeneous fibrocartilagefeatured by Saf-O-positive cartilaginous matrix in the intermediate zoneand PR-positive denser collagenous tissue in the AP bands (see e.g.,FIG. 15A). The high dose (100 mg μS/g PCL) showed likely densercartilaginous matrix in the intermediate zone as compared to low dose(50 mg μS/g PCL) (see e.g., FIG. 15A). Higher magnification imagesconsistently showed higher density of collagenous tissue in the AP bandsand fibrocartilaginous tissue in the intermediate zone withGF/μS-embedded scaffolds, in comparison to empty μS-embedded scaffolds(see e.g., FIG. 15B). Quantitatively, total collagen content in the APbands were significantly higher with the high dose as compared to lowdose of GF/μS and empty/μS (see e.g., FIG. 15C) (p<0.05; n=5 per group).Total GAGs content in the IZ were significantly higher with the highdose as compared to the low dose of GF/μS and empty/μS (see e.g., FIG.15D).

Immunofluorescence demonstrated that both high and low doses of GF/μSresulted in COL-II+/AGC+ fibrocartilaginous tissue in the intermediatezone, not in the AP bands (see e.g., FIG. 16A). No COL-II and AGC werefound with the empty μS (see e.g., FIG. 16A). Consistently, denserCOL-I+ matrix was formed with GF/μS both in high and low doses in the APbands, in comparison with the empty μS (see e.g., FIG. 16A). Relativeareas positive for COL-II and AGC were significantly wider in for thehigh dose as compared to the low dose (see e.g., FIG. 16B) (p<0.05; n=10per group). qRT-PCR showed significantly more COL-I mRNA expression withthe GF/μS for high and low doses as compared to empty μS (see e.g., FIG.16C) (p<0.05; n=5 per group).

(iv) Mechanical Properties of Engineered TMJ Discs

By 6 weeks culture with MSCs, there was no statistically significantdifference in the tensile modulus to the direction of PCL microstrandalignment between GF/μS and empty μS (see e.g., FIG. 17A). Thecompressive modulus was significantly higher in the high dose (100 mgμS/g PCL) empty μS than the low dose (50 mg μS/g PCL) both in the APbands (see e.g., FIG. 17B) and the intermediate zone (see e.g., FIG.17C). The high dose of GF/μS resulted in a significantly lowercompressive modulus, closer to that of native tissue, as compared toempty μS (see e.g., FIG. 17B-FIG. 17C).

Instantaneous (Ei) and relaxation moduli (Er) were significantly lowerin high dose GF/μS than empty μS both in the AP bands (see e.g., FIG.18A-FIG. 18B) and the intermediate zone (see e.g., FIG. 18E-FIG. 18F).Both Ei and Er in the GF/μS groups were closer to those of nativetissues as compared to the empty μS (see e.g., FIG. 18A, FIG. 18B, FIG.18E, FIG. 18F). Similarly, the ratio of Er to Ei was significantlysmaller in the high dose GF/μS as compared to all the other groups, moreapproximating the native property (see e.g., FIG. 18C, FIG. 18G).Coefficient of viscosity (μ) was significantly higher with GF/μS ascompared to empty μS both in the AP bands and the intermediate zone (seee.g., FIG. 18D, FIG. 18H). Furthermore, the high dose GF/μS showed asignificantly higher coefficient of viscosity (μ), closer to the nativelevel, in comparison with the low dose (see e.g., FIG. 18D, FIG. 18H).

Discussion.

This study's findings represent an efficient approach to engineering TMJdisc mimicking the anisotropic microstructure and heterogeneousfibrocartilage in native tissues. Anatomically correct TMJ scaffoldswere fabricated by an advanced 3D-printing technique that enablesconstruction of regionally variant microstructure and spatiotemporaldelivery of multiple growth factors. Spatiotemporal delivery of CTGF andTGFβ3 by embedding PLGA μS in PCL microstrands successfully guidedformation of native TMJ disc-like heterogeneous fibrocartilage fromMSCs. As compared to the low dose, the high dose of GF/μS in the PCLscaffold further improved the quality of the engineered TMJ disc,featured by fibrocartilaginous matrices distribution in aregion-dependent manner. In addition, the magnitudes of stress decreaseand viscosity in a stress relaxation test were also affected by the doseof GF/μS, likely demonstrating the roles of cartilaginous matrix in theviscoelastic properties of TMJ discs, consistent with other studies(Allen and Athanasiou 2006b; Willard et al. 2012). The instantaneousmodulus is determined by both elastic and viscous components, whereasthe relaxation modulus is mainly dependent on elastic component inbiological tissues. The coefficient of viscosity is the constant ofproportionality between the stress and the strain rate. Accordingly, allthe three characteristics determine the time-dependent mechanicalbehaviors of fibrocartilaginous tissues under dynamic loading conditionsthus need to be considered key criterion to evaluate engineered TMJdiscs.

The scaffolds of this study with the optimized microstructuresuccessfully resulted in the region-variant anisotropic tensileproperties. Interestingly, the tensile properties of the harvestedsamples at 6 weeks were not affected by growth factor delivery in eitherhigh or low doses of GF/μS. It is postulated that the aligned PCLmicrostrands with a higher order of tensile modulus (˜300 MPa) play adominant role in determining the tensile properties of the in vitroengineered constructs rather than newly formed ECM. In contrast, thecompressive modulus was highly affected by growth factor delivery in adose-dependent manner. After 6 weeks in vitro, MSC-seeded scaffolds withthe high dose GF/μS resulted in a compressive modulus on par with thenative level, which is significantly lower than that with empty μS.Given the higher order of magnitude of the compressive modulus of PLGAthan of PCL, the high content of PLGA μS in PCL microstrands may havecontributed to the higher compressive modulus of scaffolds. Thesignificant change in the compressive modulus of scaffoldsGF/μS-embedded scaffolds cultured with MSCs for 6 weeks is likely due tothe accelerated PLGA degradation via biochemical hydrolysis associatedwith MSC differentiation (Pan and Ding 2012). Estimated volume fractionsof PLGA μS in PCL strands from an imaging processing were 12±3.5% and23±4.2% for low and high doses, respectively. This likely provides anexplanation for the higher initial compressive and tensile moduli withhigher doses of PLGA μS. Long-term and in vivo follow-up studies willfacilitate in the understanding behind the mechanism of the acceleratedscaffold degradation related to MSC differentiation and fibrocartilageformation.

This study is primarily focused on development of TMJ disc biograft withdesign parameters for human tissues.

In conclusion, this example demonstrated a novel and efficient approachto engineering TMJ disc-like construct with heterogeneousfibrocartilaginous matrix and region-dependent viscoelastic propertiesmimicking native tissues. This study showed 3D-printed scaffolds withspatiotemporal delivery of CTGF and TGFβ3 can serve as an efficient toolin stem cell-based regeneration of TMJ discs.

Example 7: In Vitro Regeneration of Integrated Tendon-Bone Interface andLigament-Bone Interface

This example shows the successful formation or regeneration ofintegrated tendon-bone interfaces and ligament-bone interfaces withfibrocartilaginous matrix gradient mimicking native multi-tissuecomplex.

This example shows the development of micro-precise spatiotemporalgrowth factor (GF) delivery system embedded in the micro-strands of the3D printed scaffolds for integrative regeneration of multi-tissuecomplex. Temporally controlled release of the specific GFs from thespatially embedded μS in the micro-strands guide tissue specificdifferentiation of mesenchymal stem/progenitor cells.

Rotator Cuff.

A rotator cuff disorder or injury can be located in the supraspinatustendon-fibrocartilage (unmineralized and mineralized)-bone interface.˜2M Americans visit doctors every year because of a rotator cuff injury,with over 100,000 rotator cuff repairs each year. Rotator cuff repairsfailure rates can be 20% to 90% depending on the patient age, tear sizeand chronicity, muscle atrophy and degeneration, tendon quality, repairtechnique, and the postoperative rehabilitation protocol. Shoulderinjuries are frequently caused by athletic activities that involveexcessive, repetitive, overhead motion, such as swimming, tennis,pitching, and weightlifting. Injuries can also occur during everydayactivities such washing walls, hanging curtains, and gardening. 10% ofpartial-thickness tears heal and 10% become smaller, but 53% of tearswill propagate and 28% progress to full-thickness tears. Full-thicknessrotator cuff tears do not heal spontaneously, and may progress with time(Yamanaka & Matsumoto. Clin Orthop 1994, 304:68-73) (see e.g., FIG. 19).

Methods.

(i) Construction of 3D Printed Scaffolds with Spatiotemporal Delivery ofGrowth Factors

Multiple GFs, including connective tissue growth factor (CTGF),transforming growth factor β3 (TGFβ3) and bone morphogenetic growthfactor 2 (BMP2), were first encapsulated in PLGA microspheres (μS).GF-encapsulated PLGA μS were mixed with PCL slurry heated up to 100° C.and dispensed through micro-needle to construct a 3D structure using anadvanced 3D printer (Bioplotter®; EnvisionTec, Germany). Given thePLGA's melting point over 200° C. and a low heat conductivity, μS areable to maintain its original structure during the dispensing processwhile protecting bioactivities of encapsulated GFs (data not shown). Afully integrated 3D custom-designed scaffold with multiple GF-μS atdesired location was fabricated by changing dispensing cartridge duringa printing process (see e.g., FIG. 1A). Fluorescent dextran/μS was usedto confirm that PLGA μS were successfully embedded in the 3D-depositedPCL micro-strands (see e.g., FIG. 1B). The delivered GFs showed asustained release up to 42 days (see e.g., FIG. 1F).

(ii) Multi-Tissue Formation from MSCs

To confirm the bioactivities of the released GFs and its effect to guidemulti-tissue interfaces, a single layered rectangular structure wasconstructed with alternative PCL microfibers-embedding CTGF-μS andTGFβ3-μS with inter-fiber spacing of 100 μm (see e.g., FIG. 3B). Humanbone marrow derived mesenchymal stem/progenitor cells (hMSCs) (1 M/mL)were then delivered in the scaffold's inter-fiber space via fibrin gel.After 4 weeks culture, alternative depositions of COL-I+ and COL-II+matrices were demonstrated by immunolabeling. Similarly, an integratedscaffolds were fabricated comprising of three layers: PCL/CTGF μS,PCL/CTGF+TGFβ3, and PCL/BMP2 μS, for regeneration of rotator cuff(supraspinatus tendon)-to-bone interfaces between tendon,fibrocartilage, and bone (see e.g., FIG. 20 ). Another scaffold withspecified architecture and dimension was designed for ACL-to-bonecomplex formation with defined interface (see e.g., FIG. 28B). Thesescaffolds were seeded with hMSCs for 6 weeks and multi-tissue formationwas evaluated by histological analyses.

Results.

Immunofluorescence images of tendon-bone scaffolds are shown at 6 weekswith hMSCs (2 M/mL) with and without GF (see e.g., FIG. 21 ). +GF groupinduced higher collagen I expression than the −GF group (see e.g., FIG.21 , FIG. 22A-FIG. 22C). −GF group did not show any aggrecan (AGC)expression in the ECM (see e.g., FIG. 23 ). Only the BMP2 layer in the+GF group showed osteocalcin (OC) expression (see e.g., FIG. 24 -FIG. 25). The engineered tissue-bone interface shows integrated Col-I, Col-II,AGC, and OC expression mimicking the tendon-to-bone interface of nativetissue (see e.g., FIG. 26A-FIG. 26B).

Immunofluorescence images of tendon-bone scaffolds are shown at 6 weekswith hMSCs (2 M/mL) with and without GF (see e.g., FIG. 27A-FIG. 27D).Dense collagenous tissue was observed to be formed in the +GF groups. OCwas shown to be expressed in the bone and interface regions of the +GFgroups.

After 4 weeks culture with hMSCs, the scaffolds with alternativedelivery of CTGF and TGFβ3 successfully guided formation of integratedCOL-I+ and COL-II+ matrices within 100 μm interfaces (see e.g., FIG.3C). Consistently, the rotator cuff scaffolds with integrated threelayers enhanced multiphase tissue formation from MSCs by 6 weeks ascompared to scaffold without GF delivery (see e.g., FIG. 28A),demonstrated by H&E and Picrosirius Red (PR) staining. Alizarin redstaining demonstrated a localized mineralization in the BMP2μS-delivered region of the ligament-to-bone scaffolds (see e.g., FIG.28B), suggesting potential of this approach to guide spatial formationof multi-tissues in an integrated scaffold from a single population ofstem/progenitor cells.

Discussion.

The novel spatiotemporal delivery system not only enables fabrication of3D scaffolds with multiple GFs delivered at desired locations but alsocustom-designed internal microstructure and outer shape/dimension. Thesein vitro results show that spatially embedded GFs encapsulated PLGA μSfrom the scaffolds induced tissue specific differentiation of hMSCs atdifferent regions of the scaffold, which was confirmed by histologicaland immunohistochemical analyses.

This study has shown the potential of the micro-precise spatiotemporaldelivery system embedded in 3D printed scaffolds to regeneratemulti-tissue complex such as tendon-bone insertion, synovial joints,fibrocartilaginous tissues, and periodontium.

Summary.

It was shown that temporally controlled release of the specific GFs fromthe spatially embedded μS in the micro-strands guided tissue specificdifferentiation of the stem/progenitor cells, consequently leading tothe regeneration or formation of functional multi-tissue complex withintegrated interface. As such, the micro-precise spatiotemporal deliveryof GFs embedded in 3D printed scaffolds provides a novel approach foruse in multi-tissue complex formation, leading to functionalregeneration or formation of damaged or injured connective tissueinterfaces.

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1. A method of forming a biocompatible scaffold, the method comprising:(i) encapsulating at least one agent in a plurality of microspheres;(ii) combining the plurality of microspheres and a matrix material, thematrix material being suitable for forming a scaffold via 3D printing;(iii) introducing the combination of microspheres and matrix materialinto a first cartridge of a 3D printing device; (iv) heating thecombination of microspheres and matrix material in the first cartridgesufficiently to allow dispensing of the combination while preventingsubstantial degradation of the microsphere or the at least one agentencapsulated in the microsphere; (v) dispensing the heated combinationof microspheres and matrix material from the first cartridge through aprinting needle to form a polymeric microfiber, wherein the microspheresare distributed through the polymeric microfiber; and (vi) forming ascaffold comprising a plurality of the polymeric microfibers, whereinthe microspheres are distributed through the scaffold by way of thepolymeric microfibers.
 2. (canceled)
 3. (canceled)
 4. The method ofclaim 1, wherein the microspheres comprise at least a first group ofmicrospheres and a second group of microspheres; the first group ofmicrospheres and the second group of microspheres comprise at least oneagent; and the first group of microspheres and the second group ofmicrospheres comprise at least one different agent.
 5. The method offurther comprising introducing the combination of microspheres andmatrix material into a second cartridge of a 3D printing device; heatingthe combination of microspheres and matrix material in the secondcartridge sufficiently to allow dispensing of the combination ofmicrospheres and matrix material while preventing substantialdegradation of the microsphere or the agent encapsulated in themicrosphere; and interchanging the first cartridge and the secondcartridge during a printing process.
 6. The method or composition ofclaim 1, wherein the at least one agent comprises a growth factor. 7.The method of claim 1 further comprising stem cells, wherein the growthfactor stimulates fibroblastic, chondrogenic, or osteogenicdifferentiation of the stem cells.
 8. The method of claim 6, wherein afirst growth factor and a second growth factor are alternately embeddedin the microfibers.
 9. The method of claim 1, wherein the at least oneagent comprises a growth factor selected from the group consisting ofCTGF, TGFβ, TGFβ3, CTGF, BMPs, SDF, bFGF, IGF, GDF, PDGF, VEGF, or EGF,or an isoform thereof.
 10. The method of claim 1, wherein the matrixmaterial comprises polycaprolactone (PCL) and the polymeric microfibercomprises PCL.
 11. The method of claim 1, wherein the combination ofmicrospheres and matrix material are heated to less than the meltingpoint of the microsphere.
 12. The method of claim 1, wherein thecombination of microspheres and matrix material are heated to about 60°C., about 65° C., about 70° C., about 75° C., about 80° C., about 85°C., about 90° C., about 95° C., about 100° C., about 105° C., about 110°C., about 120° C., about 130° C., about 140° C., about 150° C., about160° C., about 170° C., about 180° C., or about 190° C.
 13. The methodof claim 1, wherein bioactivity of an encapsulated growth factors issubstantially maintained.
 14. The method of claim 1, wherein theprinting needle has an inner diameter of about 20 μm to about 750 μm.15. The method of claim 1, wherein the printing needle has an innerdiameter of about 50 μm to about 400 μm.
 16. The method of claim 1,wherein the 3D printed scaffold comprises microstrands having amicrostrand diameter of about 100 μm to about 400 μm.
 17. The method ofclaim 1, wherein the 3D printed scaffold comprises microstrands havingan inter-microstrand spacing or microchannel width of about 100 μm toabout 600 μm.
 18. The method of claim 1, wherein a growth factorencapsulated microsphere (μS) (i) has a diameter of about 10 μm to about600 μm; (ii) is embedded at about 10 mg to about 100 mg μS per about 1 gof matrix material; (iii) has sustained release of growth factor for atleast 42 days; or (iv) is about 50 mg μS per 1 g matrix material. 19.The method of claim 1, wherein the scaffold, composition, or polymericfiber is treated with NaOH to create micro-pores.
 20. The method ofclaim 1, wherein the method or composition results in the formation of amulti-tissue complex and the mechanical properties or composition of theresulting regenerated multi-tissue complex have substantially similarmechanical properties or composition of a corresponding nativemulti-tissue complex.
 21. The method of claim 1, further comprisingdispensing a plurality of heated matrix materials or a plurality ofcombinations heated matrix materials or microspheres from a plurality ofcartridges, the contents of each cartridge independently selected. 22.The method of claim 21, wherein each cartridge of the plurality ofcartridges (i) comprises a printing needle or a heating element or (ii)shares a printing needle or a heating element, or (iii) a combinationthereof.
 23. The method of claim 21, wherein the plurality of cartridgescomprises one or more active cartridges dispensing matrix material,microspheres, or a combination thereof.
 24. The method of claim 23,wherein the one or more active cartridges can be switched among theplurality of cartridges before, during, or after dispensing the heatedcombination.
 25. The method of claim 1, wherein heating temperature isabout the melting temperature of the matrix material and the combinationof the matrix material and microencapsulated active agent is heated suchthat 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the total volumeof microspheres do not reach more than 60° C. for more than about 10-40minutes.
 26. The method claim 25, wherein the combination of the matrixmaterial and microencapsulated active agent is heated such that 80% ofthe total volume of microspheres do not reach more than 45° C. for morethan about 30 minutes.
 27. The method of claim 21, wherein a firstprinting cartridge comprising the combination of the matrix material andmicroencapsulated active agent is switched for a second printingcartridge comprising the combination of the matrix material andmicroencapsulated active agent before 80% of the total volume ofmicrospheres do reach more than 45° C. for more than about 30 minutes.28. A method of treating a tissue defect with the scaffold producedaccording to claim 1, comprising: implanting the scaffold into a subjectin need thereof.
 29. The method of claim 28, wherein the tissue defectis associated with a multi-tissue interface selected from the groupconsisting of musculoskeletal system; craniofacial system; periodontium;cementum (CM)-periodontal ligament (PDL)-alveolar bone (AB) complex;ligament-to-bone insertion; tendon-to-bone insertion; rotator cuff;supraspinatus tendon-to-bone interface; interface between tendon,fibrocartilage, or bone; supraspinatus tendon-fibrocartilage-boneinterface; articular cartilage-to-bone junction; anterior cruciateligament (ACL)-to-bone complex; anterior cruciateligament-fibrocartilage-bone interface; intervertebral disc; nucleuspulposus-annulus fibrosus-endplates; cementum-periodontalligament-alveolar bone; muscle-to-tendon; inhomogeneous or anisotropictissues; knee meniscus; temporomandibular joint disc; periodontium;root-periodontium complex; synovial joints; or fibrocartilaginoustissues.
 30. A scaffold comprising a plurality of polymeric microfiberswherein each polymeric microfiber is embedded with a plurality ofmicrospheres encapsulating at least one agent.
 31. The scaffold of claim30, wherein each polymeric microfiber is embedded with at least a firstgroup of microspheres and a second group of microspheres; wherein thefirst group of microspheres comprises a first agent and the second groupof microspheres comprises a second agent different than the first agent.32. The scaffold of claim 30, wherein the scaffold comprises amultiphase micro-architecture and regional distribution of the firstgroup and/or second group of microspheres.
 33. The scaffold of claim 30wherein the scaffold comprises two or more layers, each layer comprisesa microfiber pattern, microfiber size or channel width that differ fromthe other